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In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin flour
Author’s Accepted Manuscript In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin flour Lyubov Dyshlyuk, Olga Babich, Alexander Prosekov, Svetlana Ivanova, Valery Pavsky, Yong Yang www.elsevier.com/locate/bcdf
PII: DOI: Reference:
S2212-6198(17)30026-8 http://dx.doi.org/10.1016/j.bcdf.2017.09.001 BCDF150
To appear in: Bioactive Carbohydrates and Dietary Fibre Received date: 28 January 2017 Revised date: 24 July 2017 Accepted date: 6 September 2017 Cite this article as: Lyubov Dyshlyuk, Olga Babich, Alexander Prosekov, Svetlana Ivanova, Valery Pavsky and Yong Yang, In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin f l o u r , Bioactive Carbohydrates and Dietary Fibre, http://dx.doi.org/10.1016/j.bcdf.2017.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin flour
Lyubov Dyshlyuka, Olga Babichb, Alexander Prosekovb, Svetlana Ivanovaa,*, Valery Pavskya, Yong Yangc a
Department of Bionanotechnology, Kemerovo Institute of Food Science and Technology
(University), 47, Stroiteley Boulevard, Kemerovo, 650056, Russia, b
Laboratory of Biocatalysis, Kemerovo State University, Krasnay Street 6, Kemerovo, 650043,
Russia c
College of Food and Bioengineering, Qiqihar University, Qiqihar, 161006, China
ABSTRACT We studied the hypocholesterolemic, antioxidant, hepatoprotective and prebiotic properties of bakery products with the content of pumpkin flour. To study the functional quality of products with pumpkin flour, in vivo studies on the influence of feed mixtures with the addition of bakery products, formulation and manufacturing technology of which were developed by us, were performed on the group of laboratory animals. After six weeks, there was a decrease in hypocholesterolemic values in serum of laboratory animals. When functional bakery products were administered in the diet of laboratory animals, there was a reduction of pathogenic and growth of lactic- and bifidobacteria in the gastrointestinal tract of the studied groups of animals. However, the activity of any enzymes studied did not reach values comparable with healthy animals.
* Corresponding author at: Kemerovo Institute of Food Science and Technology (University), Department of Bionanotechnology, Stroiteley Boulevard 47, Kemerovo, 650056, Russia. E-mail addresses: [email protected] (L. Dyshlyuk), [email protected] (O. Babich), [email protected] (A. Prosekov), [email protected] (S. Ivanova), [email protected] (V. Pavsky), [email protected] (Y. Yang) 1
Keywords: Pumpkin; Functional Foods; Confectionery Products; Antioxidant Activity; Probiotic, Hypolipidemic, Hepatoprotective Effects
Abbreviations RDA, recommended daily allowance; HDL, high-density lipoproteins; LDL, low-density lipoproteins; UFA, unesterified fatty acids; T-RFLP-analysis, terminal restriction fragment length polymorphism; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; MDA, malondialdehyde; ORAC, oxygen radicals absorption capacity.
1. Introduction
Throughout the world, diabetes, hepatitis, cardiovascular diseases and cancer are among the leading causes of death. One of the basic tenets of prevention of these and other diseases is healthy eating, and, primarily, a sufficient amount of the dietary fiber, both individually and in the form of functional foods. Products containing cereal fiber (bread with cereals, yoghurts and meat products with bran, etc.) are widely represented on the market. In addition to cereals, products of deep processing of pumpkin such as pumpkin dietary fiber and pumpkin flour are the promising source of dietary fiber (Aydin Gocmen (2015) and Ahmed, Al-Foudari, Al-Salman Almusallam (2014). Pumpkin as a plant is often regarded as the basis of functional foods and medicines. There are reports that when pumpkin powder was added to the wheat dough in the production of bread, it led not only to a satisfactory organoleptic characteristics of bread, but also increased the volume of baked loaves (Ptitchkina, Novokreschonova, Piskunova & Morris (1998); De Escalada P., Ponce, Stortz, Gerschenson Rojas (2007) and Manjula & Suneetha (2014)). Due to the presence of natural complexes of soluble and insoluble fiber, antioxidants, fatty acids, minerals, vitamins and other bioregulators, the pumpkin processing products have a wide 2
spectrum of biological activity (Rabrenović, Dimić, Novaković, Tešević Basić (2014); Nawirska-Olszańska, Biesiada, Sokół-Łętowska Kucharska (2014) and Handelman (2001)). It was previously proved that in vivo experiments, the dietary fiber from the pumpkin pulp possesses a strong antioxidant, cholesterol-lowering, hypoglycemic, hepatoprotective, and antdiabetic action (Zhao, Qian, Yin Zhou (2014); Adams et al. (2011); Xanthopoulou, Nomikos, Fragopoulou Antonopoulou (2009); Wang, Zhang Dong (2012); Makni et al. (2008); Wu, Cao Zhang (2008); Adams et al. (2011); Jun, Lee, Song Kim (2006); Barker & Syler (2009) and Simpson & Morris (2014)). The aim of study is to evaluate the medical and biological properties (antioxidant, prebiotic, cholesterol-lowering, hepatoprotective) of the bakery products with the addition of pumpkin flour in vivo.
2. Materials and methods
2.1. Materials
Wheat baking flour of premium class according to GOST R 52189-2003 (JSC Saratovsky kombinat, Russia), sugar according to GOST 21-94 (LLC "Baltiyskiy saharniy zavod", Russia), milky table margarine with fat mass fraction of 60% (LLC Moloko Zavod, Russia), salt according to GOST R 51574-2000 (LLC "Sibsol" Combinat, Russia), pumpkin flour according to TU 9146-015-70834238-10 (LLC "Victoria", Russia), functional muffins and cookies (the content of protein in 100 grams is 8.83%, fat is 3.5%, sugar is 17%, the RDA for vitamin E is 100%, P-carotene is 22.6%, magnesium is 67.8%, iron is 23 5%, zinc is 34.1%, linoleic and linolenic acids are 18.9 and 3.5%, respectively) were used in the study.
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2.2. Compilation of feed mixtures
All the components of synthetic feed mixtures (Table 1) were weighed using general purpose laboratory balance to an accuracy of 0.01 g. The base comprising saccharose, a mixture of vitamins AIN-93-VX, L-cysteine, choline, and cholesterol bitatrat was compiled and mixed for powder products with a speed of rotation of the working chamber 60 ± 5 rotations/min for 15 minutes. Then casein, a mixture of mineral salts AIN-93M, dextrinate cornstarch and powdered samples of muffins or cookies of functional purpose were added, according to the formulation and were mixed for 10 more minutes, and stirred for 15 min with starch. The obtained mixture was transferred into a homogenizer, thereto was added a solution of ghee and 2-tert-butylhydroquinone in soybean oil. According to mixture No. 1 soybean oil was also added and components were homogenized for 10 min. The obtained mixtures were stored in sealed plastic containers at a temperature of (4 ± 2)°C for not more than 5 days.
2.3. Experimental animals
Four month old male white rats (Wistar stain) weighing not less than 170±1 g (Center for Genetic Resources of Laboratory Animals of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Russia) were chosen for the study. Rats were fed a granulated complete feed (BioPro, Russia), with free access to both feed and drinking water. The duration of the quarantine before the experiment was at least 14 days. During the quarantine period the animals were placed in polycarbonate cages at a temperature of (20 ± 2) ° C and relative humidity of at least 60%. All cages with beddings, 5 animals in each cage. The cages were equipped with steel latticed lids with a stern recess, steel dividers for feed, steel label holders and drinkers for drinking water. The daily cycle of animals was 12 hours of light and dark. The experimental protocol was approved by the Local Ethical Committee of the Research 4
Institute of Biotechnology of the Kemerovo Institute of Food Science and Technology (University).
2.4. Experimental design After completion of quarantine three days before the experiment, rats were randomly divided into control and experimental groups of 10 individuals in each, and with a body weight of 170 to 200 g. The animals were housed in individual vented cages with a basement, equipped with steel lattice scaffolds with a stern recess, steel dividers for feed, drinking water drinkers, a plastic lid with a plastic label holder, removable prefilters and HEPA filters for air filtration. Cages were mounted in a rack-shelf. The air exchange rate in the system must be at least 15 m3/h. During the experiments, the following conditions were used: laboratory animals kept at a temperature of (20 ± 2)°C and relative humidity no less than 60%, the velocity of air in the system at least 15 m³/hour, duration of light cycle of 12 h/day. The animals were allowed drinking water ad libitum. The replacement of the bedding straw and drinking water was conducted daily. During the experiment, with a frequency of at least 1 time per week, the determination of body weight of the experimental animals was conducted by weighing them in a plastic beaker of 5 dm³ on laboratory scale (absolute error of ± 0.1 g). The food was replaced every day. For specimens with a weight of not more than 350 grams, the mass of daily portion of the diet was 25 grams, for specimens with a live weight of more than 350 grams was 30 grams (main nutrients). All experiments were conducted in the morning in accordance with the current recommendations for the care of laboratory animals and ethical principles of conducting painful experiments on conscious animals. Experimental animals were divided into groups in accordance with diets and experiments (Table 2). All animals were under constant observation throughout the study period.
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After the experimental period of 6 weeks, blood sampling of 5 cm³ was carried out from the cavity of the heart using sterile syringes. The position of the cardiac impulse was determined by palpation on the left side of the chest. Syringe needle was injected horizontally in relation to the sternum by 5 mm cranial to the point of cardiac impulse. The feed residues were removed from the cells 12 hours before the end of the experiment. 12 hours after the experiment, the experimental animals were placed in a turn for carbon dioxide euthanasia for 3-5 minutes, depending on the body weight of the animal. The flow rate of carbon dioxide rotameter was set at 3.5 dm³/min. The end of the exposure time of the animal in the chamber for carbon dioxide euthanasia was established visually by cessation of respiratory movements.
2.4.1. Testing of hypocholesterolemic and prebiotic properties Laboratory rats were randomly divided into three groups as follows: Group I were fed with a predominance of vegetable fats and with no additives of cholesterol (feed mixture No. 1); group II with a predominance of animal fat and addition of 0.2% cholesterol (feed mixture No. 2); group III with a predominance of animal fats, addition of 0.2% cholesterol and 15% of powdered functional bakery products (feed mixture No. 3).
2.4.2. Testing of antioxidant and hepatoprotective properties Laboratory rats were randomly divided into four groups as follows: three groups V - VII, consisting 15 animals each, and one group IV consisting of 5 animals. During the six weeks, the animals from groups IV-VI were fed with mixture No. 4, while group VII were fed with mixture No. 5. To control the initial level of biochemical parameters at the beginning of the experiment, the animals of the group IV were euthanized with blood and liver sampling. Then the animals of the group V were subcutaneously injected with sterile refined and deodorized sunflower oil at a 6
dose of 1 ml/kg body weight, using a syringe of a volume 1.0 cm³ into withers area. Animals of the group VI and VII were subcutaneously injected with oil solution of carbon tetrachloride in a dosage of 1 ml/kg of body weight, into the withers via syringe of 1.0 cm³ volume. 24, 48 and 72 hours after injection with the oil/oil solution of carbon tetrachloride to 5 animals of the groups V -VII were euthanized by carbon dioxide.
2.5. Estimation of lipid profile
Cholesterol level, triglycerides, high- and low-density lipoproteins (HDL and LDL), unesterified fatty acids (UFA) in serum samples were determined by enzyme-spectrophotometric methods (Allain, Poon, Chan, Richmond Fu (1974); Bucolo David (1973); Zilversmit Davis (1950) and Falhot, Falholt
Lund (1973)) following the manufacturer’s
recommendations using the kits “Cholesterol” (BioSystems, Spain), Triglycerides (BioSystems, Spain). Cholesterol HDL Direct (BioSystems, Spain), 96-well Serum/Plasma Fatty Acid Kit (ZenBio Inc., США). Absorbance of the solution was measured spectrophotometrically at a wavelength of 500 nm (540 nm for UFA and 600 and 700 nm for lipoprotein) in 1 cm cuvette using a spectrophotometer.
2.6. Estimation of prebiotic effect
Quantitative assessment of the composition of microbial community of fecal matter extracted from the large intestine of animals was carried out using molecular genetic method of T-RFLP-analysis (terminal restriction fragment length polymorphism) (Kitts (2001)).
2.7. Estimation of hepatoprotective properties 7
Activity of the liver indicator enzymes (LDH - lactate dehydrogenase, AST - aspartate aminotransferase (Reitman & Frankel (1957)), ALT - alanine aminotransferase (Bergmeyer, Scheibe Wahlefeld (1978) in serum was determined by spectrophotometric methods following the manufacturer’s recommendations using the kits for Lactate dehydrogenase, Aspartate aminotransferase AST/GOT, Alanine aminotransferase ALT/GPT (BioSystems, Spain), respectively. Absorbance of the solution was determined at a wavelength of 340 nm in 1 cm quartz cuvettes using a spectrophotometer with a thermostated cuvette compartment (37°C). The concentration of TBA-reactive products in serum and liver homogenates were determined spectrophotometrically in 96-well microplates at nonsorbent microplate spectrophotometer at 2 wavelengths – 535 and 572 nm and expressed equivalent concentration of malondialdehyde (MDA).
2.8. Estimation of antioxidant activity
Serum antioxidant capacity was determined by kinetic spectrophotometry (at 734 nm wavelength for 40 minutes with an interval of data collection – 1 min) by reduction in the concentration of ABTS cation radical in the reaction medium and was expressed in terms of an equivalent concentration of a water soluble analogue of vitamin E – Trolox (Cao, Sofic Prior (1997)). To calculate the values that define the ability to absorb oxygen radicals, the following equation was used: ORAC (oxygen radicals absorption capacity) = X·K·(Ssample - Sblank) / (Strolox - Sblank), where X is a sample volume (μ), K is the sample dilution factor and S is an area under the fluorescence decay curve of the sample. 8
Calculation of the sizes of the peaks and their area was performed using a software unit Fragment Analysis (Beckman Coulter, USA). For the peak identification T-RFLP-grams for the three endonucleases (HaeIII, HhaI and MspI) were processed using the program Fragment Sorter (http://www.oardc.ohio-state.edu/trflpfragsort/index.php). F8 isoprostane concentration in the blood serum of experimental animals was determined by ELISA using a commercially available kit 8-Isoprostane Express EIA KIT (Cayman Chemical, USA).
2.8. Statistical analysis
All experiments were performed three times. Data processing was carried out by standard methods of mathematical statistics. Homogeneity of the sampling effects was checked using the Student’s t-test. Differences between means are considered significant when the confidence interval is smaller than 5% (p0.05).
3. Results
Figure 1 show the results obtained from the serum samples of laboratory animals of groups I - III. After 6 weeks, the serum of the laboratory animals fed with the functional bakery products showed decrease in levels of: cholesterol by 37.8% (from 5.71 mmol/l to 3.55 mmol /l) and 21.5% (from 4.52 mmol/l to 3.55 mmol/l); triglycerides by 39.5% (from 0.76 mmol/l to 0.46 mmol/l) and 23.3% (from 0.60 mmol/l to 0.46 mmol/l); unesterified fatty acids by 30.9% (from 0.94 mmol/l to 0.65 mmol/l) and 17.7% (from 0.79 mmol/l to 0.65 mmol/l); HDL by 47.3% (from 3.47 g/l to 1.83 g/l) and 28.8% (from 2.57 g/l to 1.83 g/l) compared to the group with enhanced nutritional lipid load and without it, respectively. 9
Table 3 and Figure 2 show the results of liver indicator enzyme activity changes in the serum of laboratory animals from the group V after 24, 48 and 72 hours after administration of the oil / oil solution of carbon tetrachloride for groups V - VII, while Table 4 shows the concentration of TBA-reactive substances in the serum and liver homogenates. Subcutaneous administration of vegetable oil caused an insignificant increase in liver indicator enzyme activity of serum in laboratory animals, which cannot be said about the administration of carbon tetrachloride. It was proved that the introduction of the functional bakery products with pumpkin flour to the diet of the laboratory animals reduces the activity of liver enzymes, but it is not sufficient to bring them to the physiological standards. The useful qualities of pumpkin are well known, including its antioxidant activity, antimicrobial and hypoglycemic properties (Wang et al. (2017)). Interest is caused both by pumpkin and its derivatives (Song, Zhao, Ni & Li (2015) and Medjakovic, Hobiger, ArdjomandWoelkart, Bucar & Jungbauer (2016)), as well as the impact of processing methods on its quality (Assous, Soheir Saad & Dyab (2014); Cui & Hyuk Chang (2014); Bučko, Katona, Popović, Vaštag, Vučinić–Vasić (2015)Agrawal & Methekar (2017); Nawirska-Olszańska, Stępień & Biesiada (2017) and Wang et al. (2017)). Pumpkin seed oil also has biologically active properties (Nishimura, Ohkawara, Sato, Takeda & Nishihira (2014); Song, Ni, Hu & Li (2015); Omar & Sarhan (2017) and Al-Okbi, Mohamed, Hamed, Kassem, Mostafa (2017)). A hypolipidemic, hepatoprotective properties of pumpkin and its components are not often studied. We agree with the authors of these works that the presence, to a greater extent, of polysaccharides empowers a pumpkin and its components with these qualities. The results of the anti-atherogenic and hepatoprotective effects of a mixture of flax seeds and pumpkin on Wistar rats with a diet with 1% cholesterol are presented in the work (Maknia et al. (2008)). The authors explained the presence of these qualities by the content of unsaturated fatty acids and fibers in a mixture of seeds.
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Our results, in part, correspond to the results of work (Zhao, Qian, Yin Zhou (2014)). The authors added a powder prepared from pumpkin skin and seeds to the rats feed. Three groups of animals received a regular feed, high in fat and cholesterol, with and without powder from pumpkin. The difference in the indices (the concentration of lipids and cholesterol in the plasma of animals) of groups of animals with a normal diet and with a high fat content was 201%, 165% and 115%, respectively for TC, TG and LDL-C. The exception was HDL-C, the socalled "good" cholesterol, which fell by 16%. The addition of high-fat and cholesterol-containing pumpkin powder resulted in a 25%, 31% and 18% decrease in these parameters in plasma, respectively. HDL-C, in contrast to animals on a diet high in fat and cholesterol, increased by 16%.
4. Conclusions
Functional properties of pumpkin are well known and the study of pumpkin seed flour showed that it has equivalent properties. In this study, in vivo the hypocholesterolemic, antioxidant, hepatoprotective and prebiotic properties of experimental samples of pumpkin flour based muffins and cookies were proved which suggests use of pumpkin flour not only in bakery, but as a functional component of the formulation of other food products also. Thus, there appears the prospect of using bakery products (buns, cookies) containing pumpkin flour as an effective preventive alternative to existing analogues used in medical practice (the results of satisfactory quality and flavor characteristics of bakery products with the addition of pumpkin flour are available (Rakcejeva, Galoburda, Cude & Envija Strautniece (2011)).
Acknowledgements
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This study is based upon research supported by Foundation for Assistance to Small Innovative Enterprises in Science and Technology agreement [project number 12195r/23119].
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7
mmol/l
6 5
1
4
2
3
3
2 1 0 a
b
c
d
7
mmol/l
6 5
1
4
2
3
3
2 1 0 a
b
c
d
Fig. 1. Hypocholesterolemic value characteristics of serum samples of laboratory animals 1 group I; 2 - group II; 3 - group III (a - total cholesterol; b - triglycerides; c - unesterified fatty acid; d - high density lipoprotein). Groups I-III received the feed mixture No. 1 - 3, respectively (Table 2). The data is expressed as mean ± standard deviation (n = 15). P <0.05.
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a 700
600
U/l
500 400
V
300
VI
200
VII
100 0 24
48
72
b 100 80
V U/l
60 VI 40 VII
20 0 24
48
72
c 60 50
U/l
40
V
30
VI
20
VII
10 0 24
48
72
Fig. 2. Activity of liver indicator enzymes in the blood serum of laboratory animals of the groups V - VII after 24, 48, 72 h (a - lactate dehydrogenase, b - aspartate aminotransferase, c - alanine aminotransferase). The animals of groups V-VII were subcutaneously injected with an oil / oil solution, fodder mixtures were prepared with and without a functional additive (Table 2). The data is expressed as mean ± standard deviation (n = 15). P<0.05.
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Table 1 Composition of semisynthetic feed rations (g/kg of feed) Components Corn starch Dextrinate corn starch Casein Saccharose Soybean oil Ghee Mixture of mineral salts AINVitamin mix AIN-93-VX 93M L-cysteine Choline bitartrate Cholesterol Muffins or cookies of functional Tert – butylhydroquinone, mg purpose (milled sample)
Table 2 Experimental design Number of Group animals I 15 II 15 III 15 IV 5 V
15
VI
15
VII
15
No. 1 462.7 155.0 145.0 88.0 90.0 10.0 35.0 10.0 1.8 2.5 8.0
Feed mixture No. 2 No. 3 No. 4 460.7 310.7 510.7 155.0 155.0 155.0 145.0 145.0 145.0 88.0 88.0 90.0 10.0 10.0 50.0 90.0 90.0 35.0 35.0 35.0 10.0 10.0 10.0 1.8 1.8 1.8 2.5 2.5 2.5 2.0 2.0 150.0 8.0 8.0 8.0
No. 5 360.7 155.0 145.0 90.0 50.0 35.0 10.0 1.8 2.5 150.0 8.0
Subcutaneous administration
Feed mixture
Sterile refined and deodorized sunflower oil, 1 ml/kg of weight Oil solution of carbon tetrachloride, 1 ml/kg of weight Oil solution of carbon tetrachloride, 1 ml/kg of weight
No. 1 No. 2 No. 3 No. 4 No. 4 No. 4 No. 5
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Table 3 Activity of liver indicator enzymes in the blood serum of laboratory animals of group IV Enzyme activity aspartate alanine No. Lactate aminotransferase, aminotransferase, dehydrogenase, U/l U/l U/l 1 210.0 9.15 8.53 2 208.5 8.97 8.30 3 206.3 9.35 8.37 4 209.4 9.40 8.65 5 205.7 9.18 8.12 average* 208.0±2.4 9.21±0.21 8.39±0.25 *The data are expressed as mean ± standard deviation (n = 5).
Table 4 Concentrations of TBA-reactive substances in the blood serum, liver homogenates and blood serum oxidant capacity of laboratory animals Duration of incubation, after Group of laboratory animals subcutaneous injection of oil/oil solution of carbon tetrachloride, h IV* V VI VII TBA-reactive products, umol/dm³ MDA Control 3.30±0.15a 24 3.94±0.20a 4.22±0.21a 3.26±0.16a 48 6.84±0.34a 8.43±0.42a 3.42±0.17a 72 6.91±0.35a 11.15±0.56a 3.65±0.18a Homogenates of liver, nmol MDA/g Control 41.5±4.2b b b 24 49.8±5.0 53.2±5.3 40.8±4.1b b b 48 58.6±5.9 64.8±6.5 44.3±4.4b 72 63.4±6.3b 79.0±7.9b 46.9±4.7b Antioxidant capacity, umol TE/umol Control 5.63±0.56b b b 24 5.54±0.55 3.12±0.31 5.77±0.58b 48 5.09±0.51b 2.76±0.28b 5.34±0.53b 72 4.99±0.50b 1.99±0.20b 5.08±0.51b The data are expressed as mean ± standard deviation (n = 15 and n*=5). Values followed by same letter in a row do not differ significantly (P<0.05). 20
HIGHLIGHTS
Bakery products with the addition of pumpkin powder in vivo showed a strong antioxidant, hypocholesterolemic, hepatoprotective and prebiotic effect Bakery products with pumpkin powder are a source of biologically active components in the diet. The pumpkin bioactive ingredients and dietary fiber are determine the preventive properties of the bakery products with pumpkin
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PII: DOI: Reference:
S2212-6198(17)30026-8 http://dx.doi.org/10.1016/j.bcdf.2017.09.001 BCDF150
To appear in: Bioactive Carbohydrates and Dietary Fibre Received date: 28 January 2017 Revised date: 24 July 2017 Accepted date: 6 September 2017 Cite this article as: Lyubov Dyshlyuk, Olga Babich, Alexander Prosekov, Svetlana Ivanova, Valery Pavsky and Yong Yang, In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin f l o u r , Bioactive Carbohydrates and Dietary Fibre, http://dx.doi.org/10.1016/j.bcdf.2017.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vivo study of medical and biological properties of functional bakery products with the addition of pumpkin flour
Lyubov Dyshlyuka, Olga Babichb, Alexander Prosekovb, Svetlana Ivanovaa,*, Valery Pavskya, Yong Yangc a
Department of Bionanotechnology, Kemerovo Institute of Food Science and Technology
(University), 47, Stroiteley Boulevard, Kemerovo, 650056, Russia, b
Laboratory of Biocatalysis, Kemerovo State University, Krasnay Street 6, Kemerovo, 650043,
Russia c
College of Food and Bioengineering, Qiqihar University, Qiqihar, 161006, China
ABSTRACT We studied the hypocholesterolemic, antioxidant, hepatoprotective and prebiotic properties of bakery products with the content of pumpkin flour. To study the functional quality of products with pumpkin flour, in vivo studies on the influence of feed mixtures with the addition of bakery products, formulation and manufacturing technology of which were developed by us, were performed on the group of laboratory animals. After six weeks, there was a decrease in hypocholesterolemic values in serum of laboratory animals. When functional bakery products were administered in the diet of laboratory animals, there was a reduction of pathogenic and growth of lactic- and bifidobacteria in the gastrointestinal tract of the studied groups of animals. However, the activity of any enzymes studied did not reach values comparable with healthy animals.
* Corresponding author at: Kemerovo Institute of Food Science and Technology (University), Department of Bionanotechnology, Stroiteley Boulevard 47, Kemerovo, 650056, Russia. E-mail addresses: [email protected] (L. Dyshlyuk), [email protected] (O. Babich), [email protected] (A. Prosekov), [email protected] (S. Ivanova), [email protected] (V. Pavsky), [email protected] (Y. Yang) 1
Keywords: Pumpkin; Functional Foods; Confectionery Products; Antioxidant Activity; Probiotic, Hypolipidemic, Hepatoprotective Effects
Abbreviations RDA, recommended daily allowance; HDL, high-density lipoproteins; LDL, low-density lipoproteins; UFA, unesterified fatty acids; T-RFLP-analysis, terminal restriction fragment length polymorphism; LDH, lactate dehydrogenase; ALT, alanine aminotransferase; MDA, malondialdehyde; ORAC, oxygen radicals absorption capacity.
1. Introduction
Throughout the world, diabetes, hepatitis, cardiovascular diseases and cancer are among the leading causes of death. One of the basic tenets of prevention of these and other diseases is healthy eating, and, primarily, a sufficient amount of the dietary fiber, both individually and in the form of functional foods. Products containing cereal fiber (bread with cereals, yoghurts and meat products with bran, etc.) are widely represented on the market. In addition to cereals, products of deep processing of pumpkin such as pumpkin dietary fiber and pumpkin flour are the promising source of dietary fiber (Aydin Gocmen (2015) and Ahmed, Al-Foudari, Al-Salman Almusallam (2014). Pumpkin as a plant is often regarded as the basis of functional foods and medicines. There are reports that when pumpkin powder was added to the wheat dough in the production of bread, it led not only to a satisfactory organoleptic characteristics of bread, but also increased the volume of baked loaves (Ptitchkina, Novokreschonova, Piskunova & Morris (1998); De Escalada P., Ponce, Stortz, Gerschenson Rojas (2007) and Manjula & Suneetha (2014)). Due to the presence of natural complexes of soluble and insoluble fiber, antioxidants, fatty acids, minerals, vitamins and other bioregulators, the pumpkin processing products have a wide 2
spectrum of biological activity (Rabrenović, Dimić, Novaković, Tešević Basić (2014); Nawirska-Olszańska, Biesiada, Sokół-Łętowska Kucharska (2014) and Handelman (2001)). It was previously proved that in vivo experiments, the dietary fiber from the pumpkin pulp possesses a strong antioxidant, cholesterol-lowering, hypoglycemic, hepatoprotective, and antdiabetic action (Zhao, Qian, Yin Zhou (2014); Adams et al. (2011); Xanthopoulou, Nomikos, Fragopoulou Antonopoulou (2009); Wang, Zhang Dong (2012); Makni et al. (2008); Wu, Cao Zhang (2008); Adams et al. (2011); Jun, Lee, Song Kim (2006); Barker & Syler (2009) and Simpson & Morris (2014)). The aim of study is to evaluate the medical and biological properties (antioxidant, prebiotic, cholesterol-lowering, hepatoprotective) of the bakery products with the addition of pumpkin flour in vivo.
2. Materials and methods
2.1. Materials
Wheat baking flour of premium class according to GOST R 52189-2003 (JSC Saratovsky kombinat, Russia), sugar according to GOST 21-94 (LLC "Baltiyskiy saharniy zavod", Russia), milky table margarine with fat mass fraction of 60% (LLC Moloko Zavod, Russia), salt according to GOST R 51574-2000 (LLC "Sibsol" Combinat, Russia), pumpkin flour according to TU 9146-015-70834238-10 (LLC "Victoria", Russia), functional muffins and cookies (the content of protein in 100 grams is 8.83%, fat is 3.5%, sugar is 17%, the RDA for vitamin E is 100%, P-carotene is 22.6%, magnesium is 67.8%, iron is 23 5%, zinc is 34.1%, linoleic and linolenic acids are 18.9 and 3.5%, respectively) were used in the study.
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2.2. Compilation of feed mixtures
All the components of synthetic feed mixtures (Table 1) were weighed using general purpose laboratory balance to an accuracy of 0.01 g. The base comprising saccharose, a mixture of vitamins AIN-93-VX, L-cysteine, choline, and cholesterol bitatrat was compiled and mixed for powder products with a speed of rotation of the working chamber 60 ± 5 rotations/min for 15 minutes. Then casein, a mixture of mineral salts AIN-93M, dextrinate cornstarch and powdered samples of muffins or cookies of functional purpose were added, according to the formulation and were mixed for 10 more minutes, and stirred for 15 min with starch. The obtained mixture was transferred into a homogenizer, thereto was added a solution of ghee and 2-tert-butylhydroquinone in soybean oil. According to mixture No. 1 soybean oil was also added and components were homogenized for 10 min. The obtained mixtures were stored in sealed plastic containers at a temperature of (4 ± 2)°C for not more than 5 days.
2.3. Experimental animals
Four month old male white rats (Wistar stain) weighing not less than 170±1 g (Center for Genetic Resources of Laboratory Animals of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, Russia) were chosen for the study. Rats were fed a granulated complete feed (BioPro, Russia), with free access to both feed and drinking water. The duration of the quarantine before the experiment was at least 14 days. During the quarantine period the animals were placed in polycarbonate cages at a temperature of (20 ± 2) ° C and relative humidity of at least 60%. All cages with beddings, 5 animals in each cage. The cages were equipped with steel latticed lids with a stern recess, steel dividers for feed, steel label holders and drinkers for drinking water. The daily cycle of animals was 12 hours of light and dark. The experimental protocol was approved by the Local Ethical Committee of the Research 4
Institute of Biotechnology of the Kemerovo Institute of Food Science and Technology (University).
2.4. Experimental design After completion of quarantine three days before the experiment, rats were randomly divided into control and experimental groups of 10 individuals in each, and with a body weight of 170 to 200 g. The animals were housed in individual vented cages with a basement, equipped with steel lattice scaffolds with a stern recess, steel dividers for feed, drinking water drinkers, a plastic lid with a plastic label holder, removable prefilters and HEPA filters for air filtration. Cages were mounted in a rack-shelf. The air exchange rate in the system must be at least 15 m3/h. During the experiments, the following conditions were used: laboratory animals kept at a temperature of (20 ± 2)°C and relative humidity no less than 60%, the velocity of air in the system at least 15 m³/hour, duration of light cycle of 12 h/day. The animals were allowed drinking water ad libitum. The replacement of the bedding straw and drinking water was conducted daily. During the experiment, with a frequency of at least 1 time per week, the determination of body weight of the experimental animals was conducted by weighing them in a plastic beaker of 5 dm³ on laboratory scale (absolute error of ± 0.1 g). The food was replaced every day. For specimens with a weight of not more than 350 grams, the mass of daily portion of the diet was 25 grams, for specimens with a live weight of more than 350 grams was 30 grams (main nutrients). All experiments were conducted in the morning in accordance with the current recommendations for the care of laboratory animals and ethical principles of conducting painful experiments on conscious animals. Experimental animals were divided into groups in accordance with diets and experiments (Table 2). All animals were under constant observation throughout the study period.
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After the experimental period of 6 weeks, blood sampling of 5 cm³ was carried out from the cavity of the heart using sterile syringes. The position of the cardiac impulse was determined by palpation on the left side of the chest. Syringe needle was injected horizontally in relation to the sternum by 5 mm cranial to the point of cardiac impulse. The feed residues were removed from the cells 12 hours before the end of the experiment. 12 hours after the experiment, the experimental animals were placed in a turn for carbon dioxide euthanasia for 3-5 minutes, depending on the body weight of the animal. The flow rate of carbon dioxide rotameter was set at 3.5 dm³/min. The end of the exposure time of the animal in the chamber for carbon dioxide euthanasia was established visually by cessation of respiratory movements.
2.4.1. Testing of hypocholesterolemic and prebiotic properties Laboratory rats were randomly divided into three groups as follows: Group I were fed with a predominance of vegetable fats and with no additives of cholesterol (feed mixture No. 1); group II with a predominance of animal fat and addition of 0.2% cholesterol (feed mixture No. 2); group III with a predominance of animal fats, addition of 0.2% cholesterol and 15% of powdered functional bakery products (feed mixture No. 3).
2.4.2. Testing of antioxidant and hepatoprotective properties Laboratory rats were randomly divided into four groups as follows: three groups V - VII, consisting 15 animals each, and one group IV consisting of 5 animals. During the six weeks, the animals from groups IV-VI were fed with mixture No. 4, while group VII were fed with mixture No. 5. To control the initial level of biochemical parameters at the beginning of the experiment, the animals of the group IV were euthanized with blood and liver sampling. Then the animals of the group V were subcutaneously injected with sterile refined and deodorized sunflower oil at a 6
dose of 1 ml/kg body weight, using a syringe of a volume 1.0 cm³ into withers area. Animals of the group VI and VII were subcutaneously injected with oil solution of carbon tetrachloride in a dosage of 1 ml/kg of body weight, into the withers via syringe of 1.0 cm³ volume. 24, 48 and 72 hours after injection with the oil/oil solution of carbon tetrachloride to 5 animals of the groups V -VII were euthanized by carbon dioxide.
2.5. Estimation of lipid profile
Cholesterol level, triglycerides, high- and low-density lipoproteins (HDL and LDL), unesterified fatty acids (UFA) in serum samples were determined by enzyme-spectrophotometric methods (Allain, Poon, Chan, Richmond Fu (1974); Bucolo David (1973); Zilversmit Davis (1950) and Falhot, Falholt
Lund (1973)) following the manufacturer’s
recommendations using the kits “Cholesterol” (BioSystems, Spain), Triglycerides (BioSystems, Spain). Cholesterol HDL Direct (BioSystems, Spain), 96-well Serum/Plasma Fatty Acid Kit (ZenBio Inc., США). Absorbance of the solution was measured spectrophotometrically at a wavelength of 500 nm (540 nm for UFA and 600 and 700 nm for lipoprotein) in 1 cm cuvette using a spectrophotometer.
2.6. Estimation of prebiotic effect
Quantitative assessment of the composition of microbial community of fecal matter extracted from the large intestine of animals was carried out using molecular genetic method of T-RFLP-analysis (terminal restriction fragment length polymorphism) (Kitts (2001)).
2.7. Estimation of hepatoprotective properties 7
Activity of the liver indicator enzymes (LDH - lactate dehydrogenase, AST - aspartate aminotransferase (Reitman & Frankel (1957)), ALT - alanine aminotransferase (Bergmeyer, Scheibe Wahlefeld (1978) in serum was determined by spectrophotometric methods following the manufacturer’s recommendations using the kits for Lactate dehydrogenase, Aspartate aminotransferase AST/GOT, Alanine aminotransferase ALT/GPT (BioSystems, Spain), respectively. Absorbance of the solution was determined at a wavelength of 340 nm in 1 cm quartz cuvettes using a spectrophotometer with a thermostated cuvette compartment (37°C). The concentration of TBA-reactive products in serum and liver homogenates were determined spectrophotometrically in 96-well microplates at nonsorbent microplate spectrophotometer at 2 wavelengths – 535 and 572 nm and expressed equivalent concentration of malondialdehyde (MDA).
2.8. Estimation of antioxidant activity
Serum antioxidant capacity was determined by kinetic spectrophotometry (at 734 nm wavelength for 40 minutes with an interval of data collection – 1 min) by reduction in the concentration of ABTS cation radical in the reaction medium and was expressed in terms of an equivalent concentration of a water soluble analogue of vitamin E – Trolox (Cao, Sofic Prior (1997)). To calculate the values that define the ability to absorb oxygen radicals, the following equation was used: ORAC (oxygen radicals absorption capacity) = X·K·(Ssample - Sblank) / (Strolox - Sblank), where X is a sample volume (μ), K is the sample dilution factor and S is an area under the fluorescence decay curve of the sample. 8
Calculation of the sizes of the peaks and their area was performed using a software unit Fragment Analysis (Beckman Coulter, USA). For the peak identification T-RFLP-grams for the three endonucleases (HaeIII, HhaI and MspI) were processed using the program Fragment Sorter (http://www.oardc.ohio-state.edu/trflpfragsort/index.php). F8 isoprostane concentration in the blood serum of experimental animals was determined by ELISA using a commercially available kit 8-Isoprostane Express EIA KIT (Cayman Chemical, USA).
2.8. Statistical analysis
All experiments were performed three times. Data processing was carried out by standard methods of mathematical statistics. Homogeneity of the sampling effects was checked using the Student’s t-test. Differences between means are considered significant when the confidence interval is smaller than 5% (p0.05).
3. Results
Figure 1 show the results obtained from the serum samples of laboratory animals of groups I - III. After 6 weeks, the serum of the laboratory animals fed with the functional bakery products showed decrease in levels of: cholesterol by 37.8% (from 5.71 mmol/l to 3.55 mmol /l) and 21.5% (from 4.52 mmol/l to 3.55 mmol/l); triglycerides by 39.5% (from 0.76 mmol/l to 0.46 mmol/l) and 23.3% (from 0.60 mmol/l to 0.46 mmol/l); unesterified fatty acids by 30.9% (from 0.94 mmol/l to 0.65 mmol/l) and 17.7% (from 0.79 mmol/l to 0.65 mmol/l); HDL by 47.3% (from 3.47 g/l to 1.83 g/l) and 28.8% (from 2.57 g/l to 1.83 g/l) compared to the group with enhanced nutritional lipid load and without it, respectively. 9
Table 3 and Figure 2 show the results of liver indicator enzyme activity changes in the serum of laboratory animals from the group V after 24, 48 and 72 hours after administration of the oil / oil solution of carbon tetrachloride for groups V - VII, while Table 4 shows the concentration of TBA-reactive substances in the serum and liver homogenates. Subcutaneous administration of vegetable oil caused an insignificant increase in liver indicator enzyme activity of serum in laboratory animals, which cannot be said about the administration of carbon tetrachloride. It was proved that the introduction of the functional bakery products with pumpkin flour to the diet of the laboratory animals reduces the activity of liver enzymes, but it is not sufficient to bring them to the physiological standards. The useful qualities of pumpkin are well known, including its antioxidant activity, antimicrobial and hypoglycemic properties (Wang et al. (2017)). Interest is caused both by pumpkin and its derivatives (Song, Zhao, Ni & Li (2015) and Medjakovic, Hobiger, ArdjomandWoelkart, Bucar & Jungbauer (2016)), as well as the impact of processing methods on its quality (Assous, Soheir Saad & Dyab (2014); Cui & Hyuk Chang (2014); Bučko, Katona, Popović, Vaštag, Vučinić–Vasić (2015)Agrawal & Methekar (2017); Nawirska-Olszańska, Stępień & Biesiada (2017) and Wang et al. (2017)). Pumpkin seed oil also has biologically active properties (Nishimura, Ohkawara, Sato, Takeda & Nishihira (2014); Song, Ni, Hu & Li (2015); Omar & Sarhan (2017) and Al-Okbi, Mohamed, Hamed, Kassem, Mostafa (2017)). A hypolipidemic, hepatoprotective properties of pumpkin and its components are not often studied. We agree with the authors of these works that the presence, to a greater extent, of polysaccharides empowers a pumpkin and its components with these qualities. The results of the anti-atherogenic and hepatoprotective effects of a mixture of flax seeds and pumpkin on Wistar rats with a diet with 1% cholesterol are presented in the work (Maknia et al. (2008)). The authors explained the presence of these qualities by the content of unsaturated fatty acids and fibers in a mixture of seeds.
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Our results, in part, correspond to the results of work (Zhao, Qian, Yin Zhou (2014)). The authors added a powder prepared from pumpkin skin and seeds to the rats feed. Three groups of animals received a regular feed, high in fat and cholesterol, with and without powder from pumpkin. The difference in the indices (the concentration of lipids and cholesterol in the plasma of animals) of groups of animals with a normal diet and with a high fat content was 201%, 165% and 115%, respectively for TC, TG and LDL-C. The exception was HDL-C, the socalled "good" cholesterol, which fell by 16%. The addition of high-fat and cholesterol-containing pumpkin powder resulted in a 25%, 31% and 18% decrease in these parameters in plasma, respectively. HDL-C, in contrast to animals on a diet high in fat and cholesterol, increased by 16%.
4. Conclusions
Functional properties of pumpkin are well known and the study of pumpkin seed flour showed that it has equivalent properties. In this study, in vivo the hypocholesterolemic, antioxidant, hepatoprotective and prebiotic properties of experimental samples of pumpkin flour based muffins and cookies were proved which suggests use of pumpkin flour not only in bakery, but as a functional component of the formulation of other food products also. Thus, there appears the prospect of using bakery products (buns, cookies) containing pumpkin flour as an effective preventive alternative to existing analogues used in medical practice (the results of satisfactory quality and flavor characteristics of bakery products with the addition of pumpkin flour are available (Rakcejeva, Galoburda, Cude & Envija Strautniece (2011)).
Acknowledgements
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This study is based upon research supported by Foundation for Assistance to Small Innovative Enterprises in Science and Technology agreement [project number 12195r/23119].
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16
7
mmol/l
6 5
1
4
2
3
3
2 1 0 a
b
c
d
7
mmol/l
6 5
1
4
2
3
3
2 1 0 a
b
c
d
Fig. 1. Hypocholesterolemic value characteristics of serum samples of laboratory animals 1 group I; 2 - group II; 3 - group III (a - total cholesterol; b - triglycerides; c - unesterified fatty acid; d - high density lipoprotein). Groups I-III received the feed mixture No. 1 - 3, respectively (Table 2). The data is expressed as mean ± standard deviation (n = 15). P <0.05.
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a 700
600
U/l
500 400
V
300
VI
200
VII
100 0 24
48
72
b 100 80
V U/l
60 VI 40 VII
20 0 24
48
72
c 60 50
U/l
40
V
30
VI
20
VII
10 0 24
48
72
Fig. 2. Activity of liver indicator enzymes in the blood serum of laboratory animals of the groups V - VII after 24, 48, 72 h (a - lactate dehydrogenase, b - aspartate aminotransferase, c - alanine aminotransferase). The animals of groups V-VII were subcutaneously injected with an oil / oil solution, fodder mixtures were prepared with and without a functional additive (Table 2). The data is expressed as mean ± standard deviation (n = 15). P<0.05.
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Table 1 Composition of semisynthetic feed rations (g/kg of feed) Components Corn starch Dextrinate corn starch Casein Saccharose Soybean oil Ghee Mixture of mineral salts AINVitamin mix AIN-93-VX 93M L-cysteine Choline bitartrate Cholesterol Muffins or cookies of functional Tert – butylhydroquinone, mg purpose (milled sample)
Table 2 Experimental design Number of Group animals I 15 II 15 III 15 IV 5 V
15
VI
15
VII
15
No. 1 462.7 155.0 145.0 88.0 90.0 10.0 35.0 10.0 1.8 2.5 8.0
Feed mixture No. 2 No. 3 No. 4 460.7 310.7 510.7 155.0 155.0 155.0 145.0 145.0 145.0 88.0 88.0 90.0 10.0 10.0 50.0 90.0 90.0 35.0 35.0 35.0 10.0 10.0 10.0 1.8 1.8 1.8 2.5 2.5 2.5 2.0 2.0 150.0 8.0 8.0 8.0
No. 5 360.7 155.0 145.0 90.0 50.0 35.0 10.0 1.8 2.5 150.0 8.0
Subcutaneous administration
Feed mixture
Sterile refined and deodorized sunflower oil, 1 ml/kg of weight Oil solution of carbon tetrachloride, 1 ml/kg of weight Oil solution of carbon tetrachloride, 1 ml/kg of weight
No. 1 No. 2 No. 3 No. 4 No. 4 No. 4 No. 5
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Table 3 Activity of liver indicator enzymes in the blood serum of laboratory animals of group IV Enzyme activity aspartate alanine No. Lactate aminotransferase, aminotransferase, dehydrogenase, U/l U/l U/l 1 210.0 9.15 8.53 2 208.5 8.97 8.30 3 206.3 9.35 8.37 4 209.4 9.40 8.65 5 205.7 9.18 8.12 average* 208.0±2.4 9.21±0.21 8.39±0.25 *The data are expressed as mean ± standard deviation (n = 5).
Table 4 Concentrations of TBA-reactive substances in the blood serum, liver homogenates and blood serum oxidant capacity of laboratory animals Duration of incubation, after Group of laboratory animals subcutaneous injection of oil/oil solution of carbon tetrachloride, h IV* V VI VII TBA-reactive products, umol/dm³ MDA Control 3.30±0.15a 24 3.94±0.20a 4.22±0.21a 3.26±0.16a 48 6.84±0.34a 8.43±0.42a 3.42±0.17a 72 6.91±0.35a 11.15±0.56a 3.65±0.18a Homogenates of liver, nmol MDA/g Control 41.5±4.2b b b 24 49.8±5.0 53.2±5.3 40.8±4.1b b b 48 58.6±5.9 64.8±6.5 44.3±4.4b 72 63.4±6.3b 79.0±7.9b 46.9±4.7b Antioxidant capacity, umol TE/umol Control 5.63±0.56b b b 24 5.54±0.55 3.12±0.31 5.77±0.58b 48 5.09±0.51b 2.76±0.28b 5.34±0.53b 72 4.99±0.50b 1.99±0.20b 5.08±0.51b The data are expressed as mean ± standard deviation (n = 15 and n*=5). Values followed by same letter in a row do not differ significantly (P<0.05). 20
HIGHLIGHTS
Bakery products with the addition of pumpkin powder in vivo showed a strong antioxidant, hypocholesterolemic, hepatoprotective and prebiotic effect Bakery products with pumpkin powder are a source of biologically active components in the diet. The pumpkin bioactive ingredients and dietary fiber are determine the preventive properties of the bakery products with pumpkin
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