Article
Physicochemical properties of cookies enriched with xylooligosaccharides P Ayyappan1, A Abirami1, NA Anbuvahini1, PS Tamil Kumaran1, M Naresh1, D Malathi2 and Usha Antony1
Abstract The growing commercial importance of xylooligosaccharides is based on their beneficial health properties, particularly their ability to stimulate the growth and activity of intestinal bacteria such as Bifidobacterium and Lactobacillus species. Xylooligosaccharides are less sweet, acid, and heat stable, with low recommended levels of intake compared to other oligosaccharides. In view of the consumer demand for foods with low sugar, low fat, and high fiber contents, they are suitable for incorporation into bakery products. In this study, we have developed wheat-based cookies incorporated with xylooligosaccharides at 5%, 10%, and 15% levels. The nutritive value and physicochemical properties of the cookies changed with xylooligosaccharides incorporation; both crude fiber and dietary fiber contents increased by 14% and 35%, respectively, in the enriched cookies. The moisture levels increased with increase in the percentage of xylooligosaccharides incorporated. Cookies with 5% xylooligosaccharides were found most acceptable, although the color was slightly darker compared to the control, while cookies with 10% and 15% xylooligosaccharides were softer and darker and therefore less acceptable. Enrichment with xylooligosaccharides at 5% provided a product stable for 21 days at room temperature (25 2 C). The storage stability of cookies with higher levels of xylooligosaccharides was less than the 5% xylooligosaccharides cookies and control. The retention of the prebiotic xylooligosaccharides in the products was relatively high (74%).
Keywords Prebiotics, xylooligosaccharides, cookies, sugar replacer Date received: 29 June 2015; accepted: 15 October 2015
INTRODUCTION Lifestyle has become an important determinant of a healthy life in today’s world. Lack of physical activity, associated with overconsumption leads to nutritionrelated chronic diseases such as obesity, hypertension, cardiovascular diseases, osteoporosis, type II diabetes, and cancers (Kendall et al., 2010). Functional foods or functional food ingredients exert a beneficial effect on host health and/or reduce the risk of chronic disease beyond their nutritive value (Saarela et al., 2002; Ziemer and Gibson, 1998). Of the currently known Food Science and Technology International 22(5) 420–428 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013215617567 fst.sagepub.com
functional foods, nondigestible oligosaccharides hold an important position with respect to their prebiotic activity (Kolida and Gibson, 2007; Swennen et al., 2006). Prebiotics are defined as ‘‘non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of one or a limited number of bacterial species in the colon, such as Bifidobacteria and Lactobacilli, which have the potential to improve host health’’ (Gibson and Robertfroid, 1995). 1 Centre for Food Technology, Department of Biotechnology, Anna University, Chennai, India 2 Department of Food Science and Nutrition, Post Harvest Technology Centre, Tamil Nadu Agricultural University (TNAU), Coimbatore, India
Corresponding author: Usha Antony, Centre for Food Technology, Department of Biotechnology, Anna University, Chennai 600 025, India. Email:
[email protected];
[email protected]
Ayyappan et al. Xylooligosaccharides (XOS), a class of nondigestible food ingredients having low degree of polymerization (DP 2-6) are produced during the hydrolysis of xylan and have been generally recognized as safe by Food and Drug Association (Shandong Longlive Biotechnology Co., Ltd., Qingdao, Shandong, China, 2013). The prebiotic potential of XOS is gaining recognition in the global nutraceuticals market, though it is less exploited compared to fructooligosaccharides (FOS) and galactooligosaccharides (GOS) (Christakopoulos et al., 2003). The effect of XOS on the colonic microbiota and its metabolic activity is not as extensively studied as that of FOS and GOS (Chithra and Muralikrishna, 2010). In this context, prebiotic XOS can play an important role in the food industry as functional food ingredients. This demand has drawn researchers’ interest into extracting these ingredients from xylan-containing agricultural by-products and developing functional foods. XOS is advantageous over other nondigestible oligosaccharides in terms of both health- and technology-related properties (Vazquez et al., 2003). Depending on the type, XOS are water soluble and less sweet, typically 0.3–0.6 times of that of sucrose, and they have low daily intake (0.7–1.4 g/ day), low water activity; inhibit microbial growth, are acid and heat stable, which makes them suitable for low-calorie food preparations (Vazquez et al., 2000). XOS intake has been found highly effective for the reduction of severe constipation in pregnant women without adverse effects (Tateyama et al., 2005). With the increasing health consciousness among consumers and the rapid progress of physiologically active functional foods, the future profile of products containing oligosaccharides with biological activities seems to be greatly promising (Nakakuki, 2005). Bakery products are consumed in large quantities daily and they provide a convenient medium for delivering dietary fiber and other functional ingredients to consumers. The main challenge with adding fiber in cookies is the adverse effects on the end product quality such as color, crispiness, mouthfeel, and taste. The products with satisfying taste, palatability, and nutritional quality are in demand and have the potential to become popular in the future. Cookies refer to readyto-eat nutritive snacks containing digestive and dietary principles of vital importance, produced from unpalatable dough that is transformed into an appetizing product through the application of heat in an oven, generally containing three major ingredients; flour, sugar, and fat. They have high sugar, fat, and low final water content (1–5%) (Pareyt and Delcour, 2008). Due to their low water levels, they are microbiologically stable much longer than breads or cakes. Sucrose, a nonreducing disaccharide, is the most common sugar used in cookie preparation. It contributes to texture, flavor, sweetness, and color in cookies.
The quantity, granulation, and type of sugar used influence the quality of cookies. The aim of the investigation was to enrich cookies with XOS by partial replacement of sucrose. Laboratory scale tests with varying concentrations of XOS in cookie formulation were carried out to find the optimum level of XOS addition. Studies on the nutritive value, physicochemical properties such as dimensions (diameter, height, and weight), moisture, color, and texture as well as storage studies were carried out for the optimized cookies. The prebiotic potential of the XOS used for the enrichment of cookies was evaluated and the retention of XOS in the cookies was also estimated. The findings have potential to make available highly nutritious and functional ready-to-eat cookies for the common man.
MATERIALS AND METHODS Materials The raw materials for the cookies were purchased from the local supermarket, Chennai, Tamil Nadu, India. Commercial 95% XOS was purchased from Qingdao Oriental Tongxiang International Trading Co., Ltd., China. All the analytical grade chemicals were obtained from HiMedia and Merck (India). Methods XOS enrichment of cookies Cookie dough. The cookie dough was made with proprietary commercial formulation containing whole wheat flour (27.5%), refined wheat flour (7.0%), unsalted butter (27.5%), sugar (27.5%), salt (0.2%), sodium bicarbonate (0.2%), egg white (10.0%). Enrichment of cookies was by partial replacement of sugar with XOS at 5%, 10%, and 15%. Cookie preparation. All raw materials were weighed according to the quantity specified. Fat (unsalted butter) and sugar/XOS were mixed until fluffy. Egg white was added while mixing the flour, sodium bicarbonate (baking powder), and salt. This was added to the fat–sugar mixture and mixed for about 30 min in a dough mixer to obtain a consistent dough. The cookie dough was dropped into a round mold to form a circular shape (47 mm diameter and 4.5 mm thickness). The average weight of each cookie dough was 7.0 0.03 g. Cookies were baked at 185 C for 15–20 min and cooled. Control cookie was made with sucrose. Experimental cookies were made using three levels of XOS (5%, 10%, and 15%) as partial replacement of sucrose. Three batches of cookies were prepared and subject to analyses. 421
Food Science and Technology International 22(5) Physicochemical properties of cookies Cookie dimensions. Cookie dimensions in terms of diameter, height, and weight were measured. Diameter was measured from end to end at the center point of the cookies, while height was measured from the base to the upper surface of the center point with the help of a measuring scale. The results were expressed as millimeters (mm). The weight of cookies was determined using a weighing balance (Shimadzhu, AY 220, Tokyo, Japan) and expressed as grams (g). Diameter, height, and weight were measured in triplicates for each batch of cookies. Color measurement. The value of surface color of cookies was measured instrumentally using Hunter Lab color spectrophotometer (Ultrascan VIS, Hunter Lab, Reston, Virginia, USA). The results were expressed in terms of L*, lightness [from 0 (black) to 100 (white)], a*, greenness/redness [from a*(green) to þa*(red)] and b*, blueness/yellowness [from b*(blue) to þb*(yellow)]. The measurements were conducted under constant lighting conditions using reflectance mode at room temperature (25 2 C) with white tile as control (L*98.76, a*0.04, b*2.01). Cookies were placed in the sample holder and the reflectance was auto-recorded for the wavelength ranging from 400 nm to 700 nm and each measurement replicated five times with three cookies (triplicate). Texture analysis. A force was applied to the center of the cookie, the breaking strength and fracturability of the control and test cookies were measured by following triple beam snap method using the TA.XT plus Texture Analyzer (Stable Microsystems, Godalming, Surrey GU7 1YL, UK) with 3-point bend rig. The samples were placed on two supporting beams spread at a distance of 2.5 cm and load cell of 30 kg. The test parameters are pretest speed of 1.0 mm/s, test speed of 3.0 mm/s, posttest speed of 10.0 mm/s, distance of 5 mm, and trigger force of 50 g. Breaking strength and fracturability values were measured in each test. The breaking strength was determined from the maximum peak force (N); fracturability was determined from the distance at fracture (mm) when the cookie breaks and average values of triplicates are reported. Proximate compositions of cookies The moisture, ash, crude protein, crude fat, and crude fiber contents of cookies were determined by Association of Official Analytical Chemists (AOAC, 2000) methods in triplicates. The total carbohydrate was calculated by the difference (Southgate, 1991). The results were expressed as g/100 g cookies. Moisture was determined using the oven drying method, 5 g sample was dried in an oven at 105 C for 3 h until constant weight was achieved (925.10). Ash 422
content was determined by the incineration of a sample (5 g) in a muffle furnace at 570 C for 3 h until the ash turned gray (923.03). Crude protein was estimated by the Kjeldahl method. Total protein was calculated by multiplying the evaluated nitrogen by 6.25 (984.13). A 5 g sample was taken for fat estimation using petroleum ether (60–80 C) in a soxhlet apparatus for 5 h (922.06). Crude fiber was estimated by acid and alkali (2.5 N) digestion, 5 g of defatted sample was digested with acid and alkali followed by incineration in a muffle furnace. The final weight of the ash was calculated as crude fiber (ISO 5498-1981, reaffirmed 2010). Total dietary fiber (TDF) content in terms of soluble fiber (993.19) and insoluble fiber (991.42) of samples was determined by enzymatic method. Storage study The developed cookies were packed (each pack containing five cookies) in metalized polypropylene (PP) pouches and placed in a cupboard at room temperature (25 2 C) for 21 days. The pouches were opened and analyzed for moisture, peroxide value (PV), color, and texture on a weekly basis. All analysis of stored products were carried out in triplicates and the data compared to the fresh cookies. Moisture content The moisture content of the control and XOS-enriched cookies was determined by using the Moisture analyzer (Sartorius MA 35, Goettingen, Germany) at 105 C. One gram of cookie sample was analyzed and the results were expressed as percentage (%). Estimation of PV The fat from cookies was extracted using petroleum ether (60–80 C) for 8 h in a soxhlet apparatus. PV of the fat from the cookies was estimated by titration (AOAC, 2000, 965.33). Color and texture Color and texture of the stored cookies were estimated as described earlier. Prebioitc potential of XOS used to enrich cookies Fermentation of XOS. In order to verify the prebiotic activity of XOS used for enriching cookies, fermentation was carried out using two strains of Lactic acid bacteria (LAB) with probiotic properties. Lactobacillus acidophilus (ATCC4356) and Lactobacillus plantarum isolated in our laboratory from fermented koozh (Ilango and
Ayyappan et al. Antony, 2014) were selected for the study. The LAB cultures were maintained in de Man Rogosa and Sharpe (MRS) broth and subcultured at seven-day intervals. In vitro fermentation of XOS was done in MRS media containing the following ingredients (g/L): 10 g protease peptone, 10 g beef extract, 5 g yeast extract, 20 g dextrose/ 20 g XOS, 1 g polysorbate (Tween 80), 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulphate, 0.05 g manganese sulfate, 2 g dipotassium phosphate, with initial pH 7.0 0.2. Hundred microliters of culture suspension giving 200 colony-forming unit (CFU) was inoculated in to MRS broth (with dextrose or with XOS) and incubated at 37 C for 24 h. MRS with dextrose was taken as the blank, and MRS without dextrose and XOS was control, while MRS with XOS was the test. Growth characteristics of microorganisms was monitored by measuring the pH and absorbance (600 nm) of culture broth every 3 h. The cultures were taken out after 24 h of incubation, centrifuged (3000 g for 20 min at 25 C) and washed with distilled water twice and oven dried (85 C) to determine the dry cell mass (Chithra and Muralikrishna, 2010). Since XOS is known to be stable to heat and acid, it was assumed that destruction of XOS would be a minimal in the product and hence prebiotic efficacy of the product was not estimated. Analysis of XOS Cookies (control, XOS 5%, XOS 10%, and XOS 15%) were extracted with 80% ethanol for 6 h to remove the free sugars. To 500 mg of extracted samples, 20 mL of water was added and heated in a water bath at 90 C for 10 min. After centrifugation, the supernatant was collected and the residue was resuspended in water and heated for 30 min, which was then removed and cooled to room temperature. The supernatants were pooled, filtered, and then made upto 100 mL with distilled water. The XOS present in the extract was estimated spectrophotometrically using 3,5-dinitrosalicylic acid with xylose as standard (DuBois et al., 1956).
special preparations for health foods for elderly people and children or as active components of synbiotic preparations (Moure et al., 2006). At present the prebiotic sources like FOS and GOS are imported for use in India. A number of baby foods and dairy products are being enriched with FOS. Hence, there is a requirement to develop a larger variety of commonly consumed food products enriched with nutritional ingredients with desirable functional characteristics. In the current study, we have incorporated XOS in cookies and studied its physicochemical properties and storage stability. Physicochemical properties of cookies Dimensions of cookies. The influence of heat and the action of leavening agents are key to the changes in dimensions that occur when cookies are baked. Heating of dough leads to an increase in water vapor pressure and expansion of trapped air bubbles by approximately 1.25 times. In this case, the chemical leavening agent present (sodium bicarbonate) would have been responsible for the majority of height increase by releasing CO2. The results tabulated in Table 1 show that addition of XOS resulted in larger cookies (increase in diameter). There were not much variations in the diameter of both control and 5% XOS-enriched cookies (55.68 0.05 mm and 55.68 0.08 mm, respectively), while increasing the XOS concentration (10% and 15%) leads to significant expansion in the diameter of cookies. In contrast, the height decreased gradually with increasing addition of XOS and flattened the cookies significantly (p < 0.05). The incorporation of XOS, which has high water-binding capacity has probably caused poor gas retention leading to a decrease in cookie height. The increase in diameter and decrease in height were maximum with 15% XOS. Among the cookies containing different levels of XOS, the weight increased with the increase in XOS level from 5% to 10%, while it dropped in the 15% cookies. Therefore, the increase in weight of the
Statistical analyses Data are expressed as the mean standard deviation (SD) of three replicates. The experimental data were analyzed using analysis of variance and Duncan multiple ranges (p < 0.05). The data were analyzed according to the User’s Guide of SAS/STAT 9.1 (2004). The graphical treatment data were imported into graphics package of MS Excel.
RESULTS AND DISCUSSION There is a growing demand for prebiotic XOS as functional ingredients in foods, dietary supplements, and
Table 1. Dimensions of the XOS-enriched cookies.a Fresh cookies
Diameter (mm)
Height (mm)
Weight (g)
Control XOS 5% XOS 10% XOS 15%
55.68 0.05 55.68 0.08 55.84 0.06 56.51 0.10
5.34 0.08 5.18 0.06* 5.08 0.05* 5.01 0.10*
6.3 0.03 6.5 0.05 6.5 0.07 6.2 0.01
Note: XOS: xylooligosaccharides. a Mean standard deviation (SD) values of three determinations. *Significantly different from control (p < 0.05).
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development of color i.e., browning, in cookies is the result of two simultaneously occurring processes; the Maillard reaction where sugars interact with amino acids, and caramelization that is a direct degradation of sugars (Zanoni et al., 1995).
Color measurement Cookies with sucrose substituted by 5% XOS were slightly darker than control cookies, as demonstrated by higher L* (Lightness), a*(red color), and b* values (yellow color) (Figure 1(a)). The L*, a*, and b* values (control and XOS 5%) of cookies showed no significant changes, while there was a decrease in L* value and an increase in a* and b*values during the incorporation of 10% and 15% XOS. Significant darkening of the surface color was observed with the addition of 10% and 15% XOS (p < 0.05). This could be due to the Maillard reactions from reducing sugars in the dough, resulting in more color during baking. The
Texture analysis Texture differences were observed with respect to baked product quality (Figure 2(a)). The breaking strength and the fracturability of the XOS-enriched cookies were found to be significantly lower than the control cookies (p < 0.05), indicating a lower snapping characteristic, making the texture of the cookie soft. The cookies enriched with 5% XOS had a texture comparable to the control cookies. When XOS was substituted for sucrose, the peak force required to penetrate cookie tends to decline indicating soft nature of XOS cookies. In view of the increased height and smaller diameter,
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Figure 1. Color of XOS enriched cookies, (a) fresh cookies and (b) stored cookies.
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Ayyappan et al. the peak force was observed to be higher in case of control cookies. Gallagher et al. (2003) used raftilose for replacing sugar (20–30%) in cookies, which showed significant lower hardness levels for dough, as well as cookies (p < 0.01), suggesting softer texture characteristics. The fracturability of cookies showed no significant differences in the control and 5% XOS cookie. However, in the 10% and 15% XOS, higher values were observed, showing that, fracturabiliy increases
(a)
with the increasing levels of XOS addition; leading to cookies that are softer. Proximate composition of cookies The proximate composition of XOS-enriched cookies revealed that the moisture content increased with increased levels of XOS (Table 2) suggesting enhanced water binding. The moisture content of cookies with
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Figure 2. Texture of XOS enriched cookies, (a) fresh cookies and (b) stored cookies.
Table 2. Proximate composition of XOS enriched cookies g/100 g.a Composition of fresh cookies (%) Moisture Ash Protein Crude fat Crude fiber Total carbohydrate Total dietary fiber (soluble and insoluble)
Control
XOS 5%
XOS 10%
XOS 15%
2.74 0.61 0.54 0.02 4.90 0.45 24.30 0.77 0.24 0.09 67.28 0.38 3.13 0.04
2.81 0.04 0.57 0.05 4.98 0.19 23.87 0.54 0.62 0.01* 67.15 0.22 4.24 0.08*
3.29 0.045* 0.58 0.06 5.03 0.35 23.62 0.33 0.66 0.04* 66.82 0.29 4.87 0.05*
3.36 0.21* 0.63 0.08 5.08 0.09 23.53 0.16 0.70 0.01* 66.70 0.15 7.01 0.09*
Note: XOS: xylooligosaccharides. a Mean standard deviation (SD) values of seven estimations. *Significantly different from control (p < 0.05).
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(a) 1.8
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Figure 3. Moisture content of XOS enriched cookies, (a) fresh cookies and (b) stored cookies.
5% XOS was comparable to the control cookies, while significant increase in moisture was seen with XOS 10% and 15% (20% and 23%, respectively). The crude fiber content of XOS-enriched cookies increased to a maximum of threefold when compared to the control cookies (2.5%, 2.75%, and 2.94%). The other parameters like protein, fat, carbohydrate, and ash levels remained the same. Reports on incorporation of XOS in bakery products are limited. Handa et al. (2012) documented a seven- to ninefold increase in total fiber in cookies with partial replacement of sucrose by FOS (40%, 60%, 80%). We also observed a thirtyfive percent increase in the levels of TDF, indicating enhancement of the functional ingredient in the products. The TDF was also increased proportionally with the level of XOS addition. Storage study Many factors influence the shelf life of a product-like moisture, microbial spoilage, enzymatic changes, and oxidation (Adegoke et al., 1993). The cookies were found to be stable for 21 days when packed and stored at room temperature (25 2 C). There was no change in moisture content, color, and texture of control and 5% XOS cookies during the storage period (Figures 3, 1(b), and 2(b)). However, significant changes in moisture could be seen in the cookies with 10% and 15% XOS. No peroxides were detected in the control and XOSenriched cookies even after storage for 21 days. The color of all the cookies was retained without significant
Absorbance at 600 nm
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Figure 4. Growth characteristics of Lactic acid bacteria: (a) L. acidophilus and (b) L. plantarum.
changes during the storage period. In contrast, significant alterations in texture were observed, with higher levels of XOS causing increased softness due to moisture absorption. At the end of the storage period, the physicochemical properties of 5% XOS cookies were similar to that of control, but higher levels of 10% and 15% XOS changed the physicochemical properties significantly. Prebiotic pontential of XOS used to enrich cookies Fermentation of XOS. The ability to ferment XOS as a sole carbon source, in the MRS medium by in vitro studies was investigated. The growth kinetics for the two strains showed efficient utilization of both glucose and XOS (Figure 4). From Table 3, it is evident that pH decreased as a result of fermentation in the tested strains, although L. plantarum utilized XOS preferentially with significantly greater acidity and lower pH (5.1). The cell mass (dry weight) of L. plantarum increased rapidly and reached 10.0 mg/mL at 24 h compared to L. acidophilus with significantly lower cell mass of 6.0 mg/mL. It is clear that L. plantarum strain obtained from traditional fermented koozh can utilize XOS more efficiently. Ananieva et al. (2012) studied the utilization of XOS by different Lactobacillus strains like Lactobacillus plantarum, Lactobacillus brevis, and
Ayyappan et al. Table 3. Growth of lactic acid bacteria with XOS as carbon source.a Lactic acid bacteria (LAB)
Control (MRS with dextrose)
XOS (MRS without dextrose)
Absorbance Cell mass Absorbance Cell mass (600 nm, 24 h) pH (7 0.2) (mg/mL broth) (600 nm, 24 h) pH (7 0.2) (mg/mL broth) L. acidophilus (ATCC4356) 1.45 0.05 L. plantarum (Lab isolate) 1.86 0.1
5.9 0.33 5.5 0.31
5.8 0.48 8.2 0.31
1.62 0.1* 1.98 0.05*
5.8 0.25 5.1 0.22*
6.0 0.52 10.0 0.5*
Note: XOS: xylooligosaccharides. a Mean standard deviation (SD) values of two lactic acid bacteria. *Significantly different from control (p < 0.05).
Lactobacillus sakei. It was observed that L. plantarum showed similar specific growth rates on glucose and XOS; L. brevis had relatively low growth rate on XOS, while L. sakei did not utilize XOS, which could be due to its homofermentative property, suggesting that the utilization of XOS was strain specific. The results here confirm the prebiotic activity of XOS used for the production of cookies. Analysis of XOS The extraction of residual XOS remaining in the cookies after baking is necessary to quantify its retention in the product. The retention of XOS in food products has not been reported in literature. In our study, we observed 73.8% retention of XOS in cookies enriched at 5% level. The XOS-added cookie (5%) had good binding and stability and was almost equivalent in its physicochemical properties and storage to the control cookie. Stability and retention of the functional ingredient are fundamental for deriving the associated health benefits (Wang and Gibson, 1993). Therfore, it would be reasonable to assume that the prebiotic activity of the XOS is retained substantially in the product.
CONCLUSION The present study confirms the potential of producing functional food products containing XOS, which has been demonstrated with cookies. Partial replacement of sucrose with 5% XOS into the cookie formulation did not change the physicochemical properties of the cookies, but increased the crude fiber content threefold and dietary fiber by thirtyfive percent. The enriched cookies retained 74% of the incorporated XOS, indicating the prebiotic functionality of the product. Each enriched 5% XOS-enriched cookie contains on an average 0.1 g XOS and consumption of 8–10 cookies per day is sufficient to meet the recommented intake of XOS (0.7–1.4 g/day). In addition, incorporation of XOS at 5% gave a product that was shelf stable for a period of 21 days at room temperature (25 2 C) in PP
pouches. The present study gives a promising lead to further exploit XOS as prebiotic source to be incorporated in other functional food products. However, it is neccessary to confirm the functionality of XOS in the enriched cookies in vivo. DECLARATION OF CONFLICTING INTERESTS The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
FUNDING The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors wish to thank Science and Engineering Research Board/Ministry of Food Processing Industries, Government of India for funding this research grant under the R & D project: No. SERB/MOFPI/0034—2012 (AU).
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