Sensory properties, physical and microbiological studies of pumpkin seed (Cucurbita pepo) blended cakes

Sensory properties, physical and microbiological studies of pumpkin seed (Cucurbita pepo) blended cakes were evaluated using standard analytical and microbiological methods. Peroxide values of blended cakes were also evaluated. CF2 blend (20% pumpkin) had the highest mean sensory scores for all sensory parameters and was found to be higher than control (WF1) with overall acceptability of 8.25±0.58/ 5.36±0.61 at day 1/ day 4 and 8.40±0.64/ 8.20±0.96 at day 1/ day 4 under ambient and refrigeration conditions respectively. The weight (g) of the blended cakes ranged from 34.95±0.27 to 39.16±0.02. CF6 (60% pumpkin) cake recorded the highest weight of 39.16±0.02 g while wheat flour (WF1) cake recorded the least weight of 34.95±0.27 g. The volume (cm3) and specific volume (cm3/g) of blended cakes ranged from 91.89±0.17 to 96.61±0.07 and 2.35±0.04 to 2.75±0.14 respectively. Peroxide values of blended cakes ranged from 4.23±0.03 meq/kg to 4.92±0.04 meq/kg. CF2 cake had the highest peroxide value of 4.92±0.04 meq/kg among the blends, although lower than that of WF1 cake while CF6 cake had the least peroxide value of 4.23±0.03 meq/kg. Bacterial and fungal counts (CFU/g) of baked cakes ranged from 1.40x102±0.58 to 2.99x103±1.00 and 2.50x101±1.00 to 2.40x102±2.08 respectively. WF1 cake recorded the highest bacterial and fungal counts (CFU/g), while CF6 recorded the least bacterial and fungal counts (CFU/g) for both storage days under ambient and refrigeration conditions. From the results obtained, 20% supplementation of pumpkin seed flour into wheat flour (CF2) produced cakes with the most acceptable sensory, physical and microbiological qualities, when compared with control and other levels of blends. Therefore, there is need for inclusion of pumpkin seed flour (PSF) into wheat flour (WF1) in order to enhance the shelf life stability, nutritional properties, quality and acceptability potentials of baked cakes.


Introduction
Pumpkin belongs to the family Cucurbitaceae. It is a plant that has been traditionally used as a medicine in developing countries and obtained revival of use in the United States and Europe [1]. Pumpkin has gained extensive attention in current times due to the good nutritional composition and health shielding values of its seeds [2]. Pumpkin seeds are loaded with nutrients and medicinal properties, due to which the seeds are used for remedial purposes all over the world. Pumpkin seeds are often eaten as snack after roasting and salting in Arab countries [3].

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Proteins are commonly employed as food ingredients on the basis of their importance in human diets. The best proteins belong to animal sources, since they are nutritionally better. However the high cost of animal protein makes vegetable protein the main dietary component for most of the world's population. The use of plant protein in the formulation of new food products or in conventional foods has been the focus of most research in recent years. One gram of pumpkin seed protein contains as much tryptophan as a full glass of milk. The main storage proteins are salt soluble globulins accompanied by glutelins and lesser amounts of albumins and prolamins [4].
Pumpkin is still not widely used in the food industry, but is consumed in homes worldwide in dishes such as pumpkin bread, soup, pie, etc [5]. The hulled or semi-hulled seeds of pumpkins could be roasted and eaten as snack. The seed can be prepared for eating by first separating them from the pumpkin flesh, then coating them in a generally salty sauce after which the seeds are distributed upon a baking sheet, and then cooked in an oven at a relatively low temperature for a long period of time [6; 7].
Pumpkin (Cucurbita pepo) has received attention in recent years because of the nutritional and health protective values of the seeds [8]. The seed is an excellent source of protein which also has pharmacological activities such as antidiabetics [9], anti-fungi [10], antibacterial and anti-inflammation activities [1]. Research has shown that the seeds have antioxidant effects [11]. Proteins may contribute to food flavour through one of the following: bound molecules, absorption, breakdown products and chemical reactions. In addition to the good nutritional and health benefits of Cucurbita seeds, they are cheap and are widely distributed [12].
The serious consequences of malnutrition particularly among infants and children form a primary roadblock to social and economic development. Recently, more attention has been focused on the utilization of underutilized agricultural products. Obviously such utilization would contribute to maximizing available resources and results in the production of various new food products. Preservation of vegetable and fruits is an excellent way to curb post-harvest losses which are major challenges in sub-Sahara African countries [13; 14].
Processing methods are employed mostly to preserve and improve the organoleptic properties of foods, microbial quality and equally to enhance shelf life [15]. The rich nutrition base of pumpkin can be tapped to improve the nutritional quality of baked products, soup and sauces [13]. A pleasing green colour and nutty taste of pumpkin seed flour makes it feasible to be used in developing new food products such as in fortified wheat flour to produce bakery products like pastries with unique and nutty taste [16]. The use of white flour from processed whole wheat grain leads to drastic reduction in nutritional density and fibre content [17]. These days, awareness by consumers on the need to eat high quality and healthy foods known as functional foods that contains ingredients providing additional healthy benefits beyond the basic nutritional requirements, is increasing [18]. Hence, this work is aimed at evaluating the sensory properties, physical and microbiological studies of pumpkin seed (Cucurbita pepo) blended cakes.

Sensory evaluation
Twenty five (25) samples of the cake were prepared using wheat flour (WF1) as control, and composite samples prepared by supplementing wheat flour with pumpkin seed flour at different percentages (20%, 40%, 50% and 60%) representing CF2, CF4, CF5 and CF6 respectively. Sensory evaluation test was done on the first day of production and the fourth day during storage at both ambient and refrigeration conditions. The consumer acceptance of five different samples of cakes was evaluated by twenty five judges comprising untrained candidates. The sensory evaluation test was conducted in the air-conditioned laboratory, which provided a quiet and comfortable environment. The cakes were served on white disposable plastic trays and packaged-potable water was provided for mouth rinsing. Samples were coded with different symbols and the sample order was randomized. The judges were asked to evaluate the colour, taste, flavor, texture and overall acceptability of the cakes using a 9-point hedonic scale.

Physical characteristics of the cakes
The physical characteristics of the enriched cakes were measured using the method outlined by [19] and reported by [20] for fluted pumpkin cookies and adopted by [21].

Peroxide value determination
The method described by [22] was adopted in the determination of peroxide value of pumpkin (Cucurbita pepo) seed composite flours.

Microbiological analyses of baked cakes
Baked cakes from composite flours were analyzed for bacterial and fungal counts using spread plate method as described by [23]. Ten fold serial dilutions of flour samples were done using sterile peptone water as diluent. One gramme (1 g) each of mashed sample was aseptically transferred into a sterile test tube containing nine milliliter (9 mL) of sterile peptone water, stirred with sterile glass rod and was shaken vigorously to ensure adequate disengagement of microorganisms to obtain 10 -1 dilution. Serial dilutions of the homogenates were continued and made step-wisely till the fifth (5th) tube, to obtain dilutions of 10 -2 to 10 -5 . Spread plate techniques of [23] were used to enumerate bacteria and fungi in the samples and each dilution was plated in replicates using plate count agar for mean bacterial count, and fortified sabouraud dextrose agar (SDA) for mean fungal count. The plates were incubated at 35±2 °C for 72 hours for mean bacterial counts and 25±2 °C for 120 hours for mean fungal counts. Pure bacterial isolates were identified using cultural, morphological and biochemical characterization. Identification of the bacteria to genera level was based on the schemes of [24]. The purified fungal isolates were identified on the basis of macroscopic and microscopic characteristics by slide culture technique and lactophenol staining. The scheme of [25] was used for the identification.

Statistical analyses
All obtained data in this study were analyzed using analysis of variance (ANOVA). Descriptive statistics in form of mean, standard deviation and Duncan post hoc were also used to assess the data, and analyses were done using SPSS version 20 (Statistical Product and Service Solutions).

Sensory evaluation
The mean scores of sensory evaluation of cakes by semi-trained panelist using nine-point hedonic rating scale for day 1 and day 4, under ambient and refrigeration conditions are presented in Table 1. The highest scores for all sensory parameters was obtained by CF2 blend and was found to be higher than control (WF1) with overall acceptability of 8

Physical properties
The physical properties of baked cakes are shown in Table 2. The weight (g) of the cakes from the blends ranged from 34.95±0.27 to 39.16±0.02. CF6 cake recorded the highest weight of 39.16±0.02 g while WF1 cake recorded the least weight of 34.95±0.27 g. The volume (cm 3 ) and specific volume (cm 3 /g) of cakes from blends ranged from 91.89±0.17 to 96.61±0.07 and 2.35±0.04 to 2.75±0.14 respectively. CF2 cake recorded the highest volume and specific volume of 96.61±0.07 cm 3 and 2.75±0.14 cm 3 /g, while CF6 cake recorded the least volume and specific volume of 91.89±0.17 cm 3 and 2.35±0.04 cm 3 /g respectively. Values obtained when compared were statistically significant (p<0.05).

Peroxide value
The results of peroxide values of blended cakes are presented in Table 3. Peroxide values of blended cakes ranged from 4.23±0.03 meq/kg to 4.92±0.04 meq/kg. CF2 cake had the highest peroxide value of 4.92±0.04 meq/kg among the blends, although lower than that of WF1 cake while CF6 cake had the least peroxide value of 4.23±0.03 meq/kg ( Table  3). All values obtained were statistically significant (p<0.05).

Microbiological analysis of cakes
The microbial qualities of baked cakes at first and fourth days under ambient and refrigeration conditions are shown in  4. Discussion

Sensory evaluation
The highest score for all sensory parameters was obtained by CF2 blend and was found to be higher than control. The overall acceptability score of CF2 blend was significant (p<0.05) and higher than that of control and other three blends. Thus, indicating that cake supplemented with pumpkin seed flour at 20% was better accepted than control (WF1) or any other blends. The obtained sensory result in this study is in agreement with the report of [2] during a work with cake supplemented with raw pumpkin seed flour involving four samples viz-a-viz one control and three test samples of different supplemented levels (10%, 20% and 30%) and the overall acceptability score of T2 (20% pumpkin) treatment in that research was higher than that of control and other two treatments. Thus, indicating that the cake supplemented with raw pumpkin seed flour at 20% level was better accepted than cake prepared with refined wheat flour. The sensory scores of day 1 were better and higher than that of day 4.
Consequently, sensory scores of cake samples kept under refrigeration were better and higher than that stored under ambient conditions. Notwithstanding, a general observation indicted pumpkin flour with an after-bitter taste in substitution percentages above 20 in the blended cakes, which intensified as storage days prolonged, hence the poor recorded scores in taste parameter ( Table 1) . The poor sensory scores at day 4 especially for samples stored under ambient conditions could also point towards spoilage of the cake. This is an indication of deterioration of physical, chemical and organoleptic properties of stored cake owing to microbial activities, which eventually causes spoilage of the cake. Hence, with the poor overall acceptability scores, there is need for preservatives and better storage conditions in order to extend the shelf life of the stored cake beyond 4 days.
Also, the addition of pumpkin seed flour affected the appearance, color, taste, texture, flavour and overall acceptability of the cakes. [26] reported that the different results in sensory attributes of pumpkin blended cakes may be due to the color and stronger flavor of pumpkin powder. [27] clarified that adding pumpkin powder to the cake enhanced the flavor of the cake. [28] stated that significant difference in sensory attributes exists between 10% and 15% pumpkin powder bread. [29] reported that the sensory characteristics of spongy cake processed using 5%, 10% and 15% of pumpkin meal were acceptable to most members regarding to taste, odor, texture, color, appearance and overall acceptability. Also, [30] during the evaluation of physico-chemical properties of biscuits supplemented with pumpkin seeds to fight malnutrition in children of Pakistan reported that substitution treatment with 15% pumpkin seed flour scored highest (8.0) for maximum overall acceptability and an increasing trend in all sensory attributes among various treatments when compared with control.

Physical properties
As the blending ratio of PSF into WF1 increased, higher was the weight of the baked cakes and lower was the volume and specific volumes of the baked cakes. Although with CF2, there was a sharp increase of volume and specific volume, higher than that obtained with WF1, which later decreased with substitution of WF1 with higher ratios of PSF. This substitution of WF1 with increasing ratios of PSF resulted in significant increase in weight and decrease in volume and specific volume of baked cakes (p<0.05).
Cake weights significantly (p<0.05) increased with increasing ratio of PSF (Table 2). In a similar report, [28] illustrated that increasing levels of pumpkin flour in bread manufacture (0-15%) significantly increased the weight of loaf among samples and attributed this increase in weight to the pumpkin fiber content.
With increasing pumpkin flour ratios, volume and specific volumes of cakes decreased significantly (p<0.05). According to [28], reducing the amount of wheat flour in the bread formulation caused less gluten matrix, resulting in bread with lower specific volume compared to the control. Also, it was stated that substitution of more than 10% pumpkin flour resulted in lower loaf volume of bread as compared with the control. A similar result to the assertion by [28], was recorded in this study in Table 2 with CF2. The less gluten matrix in pumpkin flour might cause a lower volume and specific volume of cake. Also, the decreased cakes volume and specific volume recorded in Table 2 among blends could be due to lower gas retention, causing less expansion of the product which is attributable to lower peak viscosity of flour [31]. Weights and volumes of baked products have been reported to be dependent on bulk density of the flour blends used [32]. The results obtained in the study are in agreement with the reports of [28; 32; 33].

Peroxide value
WF1 cake which was the control had the highest peroxide value when compared with the blended cakes. Conclusively, substitution of WF1 with different ratios of PSF during cake making resulted in significant (p<0.05) decrease in peroxide values among blended cakes. As the blending ratio of PSF into WF1 increased, lower was the peroxide value of blended cakes. Hence, the substitution of WF1 with increasing levels of PSF resulted in significant (p<0.05) decrease in peroxide values among blended cakes as shown in Table 3.
Peroxide values obtained in the blended cakes were retrogressive with increasing level of additions of PSF into WF1. Peroxide values of the blended cakes decreased as the level of PSF substitution increased. The decrease in peroxide values among the blended cakes may be attributed to high oil absorption capacity of wheat flour resulting from high starch content during baking [31].
Results of peroxide values obtained in this study were statistically significant (p<0.05) and correlated with the assertions of [2]. According to [34] more peroxide value is harmful for shelf life of products. From the results obtained, blended cakes had a decreased peroxide values among the various baked cakes, which implies that substitution of WF1 with increasing levels of PSF during baking reduces the chances of rancidity and increases the shelf life of products.

Microbiological studies
There were significant differences (p<0.05) among various samples when compared along storage days. With the obtained high microbial counts recorded at day 4 especially during ambient storage condition, coupled with deteriorated sensory results obtained at same day 4, there are indications of poor storage-quality condition of the cake leading to spoilage.
It was observed that WFI had higher bacterial and fungal counts at both day 1 and day 4 than the blends. Cakes stored under refrigeration had lower microbial counts compared to those stored under ambient conditions. The chilling of the refrigerator affected adversely the replication of microbial cells. This gave rise to reduced growth rate and counts observed among samples stored under refrigeration over those stored under ambient conditions. This confirms the assertion of [35] that most food spoilage microbes fall into the mesophilic group. Also observed during the study, was the correlation between moisture content, blending ratios and microbial counts [31]. As the moisture content decreased, lower were the recorded microbial counts. Also, as the blending ratio increased, lower were the recorded microbial counts. The former could be as result of the low moisture content of PSF over WF1, while the later could be attributed to high oil content of pumpkin seed flour (PSF) over wheat flour (WF1) as was observed by [31]. The results obtained in this study are in agreement with the works of [2] and [36].
Oil is an inhibitory substance that limits growth of aerobic microorganisms. It functions by sealing up the air pores through which air could flow in to support growth of aerobic microorganisms, thereby creating an unconducive environment that is devoid of oxygen (anaerobic). Oil on the other hand, could be used in preserving food and other edible items because of its negative tendencies in supporting microbial growth especially the aerobes [35].
The low moisture content of PSF over WF1 was another factor pointing towards the low microbial counts recorded in the blends. Both moisture content and microbial counts are directly proportional as the lower the moisture content, the lower was the microbial counts. According to [37], the preservation of foods by drying is a direct consequence of removal or binding of moisture, without which microorganisms do not grow. Moisture content could also be described in terms of water activity (aw) which is referred to as water requirements of microorganisms in the environment. This concept is related to relative humidity (RH) as shown: RH = 100 × aw. Water activity plays a role with moisture content in the growth of microorganisms, as it is the available water for microbial growth.

Conclusion
The ravaging consequences of malnutrition among infants and children; preservation of vegetable and fruits as an excellent way to curb post-harvest losses which have been a major challenges in sub-sahara African countries; and utilization of underutilized agricultural products have resulted in the production of products with improved nutritional contents that could bridge the nutritional requirements lacking among infants and children. From the recorded results, 20% supplementation of pumpkin seed flour into wheat flour (CF2) produced cakes that had the most acceptable sensory, physical and microbiological qualities when compared with control and other levels of blends. Inclusion of pumpkin seed flour into baking processes enhances the organoleptic characteristics, shelf life properties and nutritional potentials of the baked cakes. Therefore, there is need for inclusion of pumpkin seed flour (PSF) into wheat flour (WF1) in order to improve the nutrient content, quality and acceptability potentials of our baked products.