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Abstracts of International Conference on

Advances in Human Nutrition, Food Science & Technology 2016

Toronto, Canada

26-27 June 2016

ABSTRACTS

Unique Conferences Canada Publication Toronto, Canada

Abstracts of International Conference on Advances in Human Nutrition, Food Science & Technology 2016

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Published by

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Printed in Canada

June 2016

ISBN 978-0-9951985-1-7

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NutriFood2016

CONFERENCE CONVENER

Prabhath Patabendi


CONFERENCE CHAIR

Dr. Ashish Joshi

City University of New York School of Public Health

HOSTING ORGANIZATION


INTERNATIONAL SCIENTIFIC COMMITTEE

Dr. Ying Zhang ( Australia)

Prof. Ian Newman (USA)

Prof. Ananda Kumara (Japan)

Prof. Mahanama De Zoysa( South Korea)

Prof. S.L. Ranamukarchchi( Thailand)

Dr. Marie Caesarini( Indonesia)

Prof. Ashish Joshi (USA)

Dr. Premachandra Wattege( UK)

Dr. Shashi Kant (USA)

Dr. Minika Nitschke (Australia)

Dr. Kevin Morford (UK )


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TABLE OF CONTENTS

Abstract Titles Presenters Page No

1 Investigation into the inhibitory effects of tea consumption on iron bioavailability in a cohort of healthy UK women, using a stable iron isotope (57Fe)

Salma Faeza AHMAD FUZI

9

2 Plasma Cholesterol, Heart Disease and Functional Foods Zhen-Yu Chen 10

3 Study of process conditions for obtaining black soymilk Thiana ESTEVES 11

4 Influence of Maternal Nutritional Status on Pregnancy Outcomes Parmeet Kaur

12

5

Efficacy of fish oil-derived n-3 polyunsaturated fatty acids on inflammatory and oxidative stress markers in a randomized controlled trial of type 2 diabetes mellitus patients with abdominal obesity

Feng Wang 13

6 An Empirical Analysis Of The Death Of Children Under Five Years In Rwanda

Fidence Munyamahoro 14

7 Antineoplastic activity of the essential oils from Chrysanthemum morifolium Ramat

Guiju Sun 15

8 Hypolipidemic effects of n-3 PUFA on Type 2 Diabetes with hyperlipidemia in postmenopausal women

Hui Xia 16

9 Effect of partially hydrolyzed guar gum (PHGG) on postprandial hyperglycemia, hyperlipidemia and incretins metabolic hormones in healthy and glucose intolerant subjects

Mahendra P. Kapoor 17

10 Development of a post-fermented Tea and its Physiological Effects

Kieko Saito 18

11 “Pan de muerto”, Mexican traditional bread: Understanding its characteristics and staling by relating its fat content to starch retrogradation

Anne MATIGNON 19

12 The micro-nutrient Composition of Chloroplasts from Range of Green Leafy Tissues

Mohamed A. Gedi 20

13 Increasing Broiler Carcass Yield by Protein and Methionine Supplementation

Abbas Mohammadpour 21

14 Effects of Atmospheric radio-frequency (RF) Plasma on Mold Spoilage, Color, Nutritional Value, and Mineral Content of the Whole Grain Brown Rice

Narumol Matan 22

15 Evaluation of Potentials of some selected seeds’ flours as partial Substitute for Wheat in Cookies production

Omobolanle Olorode 23

16 Effect of a beverage made of andean grains, quinoa, kaniwa and kiwicha in the metabolic control of Obese Patients with Diabetes

D.Patricia Garrido 24


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17 Association of body composition and sleep behaviours in young Saudi females: A cross sectional survey

Syeda Fatima Bushra 25

18 Simultaneous Determination of Six Kinds of Nutritional & Functional Ingredients In Fresh Citrus Fruit by RP-HPLC-DAD Wavelength Switching Method

Weisheng Xu 26

19 Evaluation of Waist Circumference, Waist/Hip Ratio, Waist/Height Ratio and Human Body Shape Index (HBSI) of University Students

Sultan Nazif 27

20 Body Composition of Soccer Referees in the Turkish Republic of Northern Cyprus

Fatma Hülyam Eren 28

21

The Effects of Ginseng Extracts on Perceived Stress, Blood Pressure, Bi-spectral index and Pulse Oxygen Saturation in Normotensive healthy adults after exposure to white noise stressor

Myung-Haeng Hur 29

22 TIgE level and IgE-mediated allergy of 3-12 years urban children in China

Lichen Yang 30

23 Control of Salmonella Enteritidis on fresh beef cuts with soy edible coatings containing thyme and oregano essential oils

Gökçe Polat Yemiş 31

24 Haze Formation in Clarified Pomegranate Juice and Concentrate Oktay Yemiş 32

25 Hypoglycemic, Hypolipidemic and Antioxidant Activities of Musa Paradisiaca, Normalis (Plantain) Supplemented Diet on Alloxan Induced- Diabetic Albino Rats

Friday O. Uhegbu 33

26 Food and nutrition data in the Wolfram Language Andrew Steinacher 34

27 Soybean Meal Extracts as Alternative to Antibiotics Use in Enteric Infections Control

Jenifer Mota Rodrigues 35

28 The Use of Pure and Commercial Pepsins in the Obtainment of Antihypertensive Whey Peptides

Caroline Melliger-Silva 36

29 Proteome Approach to Characterize Proteins Induced in Strawberry Leaves after Salicylic Acid Treatment

Caroline Melliger-Silva 37

Paper Titles Submitting Authors

i Study of process conditions for obtaining black soymilk Thiana ESTEVES 41

ii An Empirical Analysis Of The Death Of Children Under Five Years In Rwanda

Fidence Munyamahoro 47

iii

The Effects of Ginseng Extracts on Perceived Stress, Blood Pressure, Bi-spectral index and Pulse Oxygen Saturation in Normotensive healthy adults after exposure to white noise stressor

Myung-Haeng Hur 55

iv Soybean Meal Extracts as Alternative to Antibiotics Use in Enteric Infections Control

Jenifer Mota Rodrigues 63


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ABSTRACTS


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Investigation into the inhibitory effects of tea consumption on iron bioavailability in a

cohort of healthy UK women, using a stable iron isotope (57Fe)

Salma Faeza, AHMAD FUZI1 2

; Sohail, MUSHTAQ1

1Department of Clinical Sciences & Nutrition, University of Chester, United Kingdom

2Department of Nutrition & Dietetics, Universiti Putra Malaysia, Malaysia

Tea can inhibit iron absorption but it is unclear whether time of consumption in relation to a meal

influences bioavailability. The present study assessed the effect of time variability of tea consumption

on iron absorption from a test meal in non-pregnant women using a 57

Fe tracer. Twelve women

(mean age (± SD): 24.8 ± 6.9 years) were given a standardised breakfast extrinsically labelled with

4mg 57

Fe on 3 occasions with a 14 day time interval between each test meal. The isotope was

administered with: (a) water (b) tea simultaneously (c) tea 1 hour after the meal. A reference dose

was also administered (3mg 57

Fe with 35mg ascorbate) without a test meal. Fasted blood samples

were collected and iron absorption was estimated by the erythrocyte iron incorporation method,

assuming 80% absorption. Mean iron absorption (± SD) was 5.69 ± 8.5%, 3.57 ± 4.2% and 5.73 ±

5.4% (test meals a,b and c, respectively). Mean iron absorption was found to be 2.2% higher when

tea was consumed 1 hour after test meals (p = 0.05). Mean total iron absorbed was also found to be

0.043mg higher when tea was consumed 1 hour after the meal (p= 0.049). This study demonstrates

that consuming tea separately from a meal may attenuate the inhibition effect thus increasing iron

absorption. This may have implications for those who are iron deficient.

Keywords: Iron deficiency; Iron bioavailability; Tea


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Plasma Cholesterol, Heart Disease and Functional Foods

Zhen-Yu Chen

School of Life Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China

Cholesterol is always an important issue because plasma total cholesterol (TC) and low-density

lipoprotein (LDL) correlate strongly with the risk of coronary heart disease. Cholesterol homeostasis is

maintained by a complex mechanism which involves the sterol absorption, anabolism, catabolism and

excretion. Nutraceuticals and functional foods which lower plasma TC can affect the genes which

regulate cholesterol homeostasis. In general, cholesterol-lowering functional foods and nutraceuticals can

be classified into seven types namely intestinal Niemann-Pick C1 like 1 (NPC1L1) competitors, intestinal

acyl-CoA: cholesterol acyltransferase 2 (ACAT2) inhibitors, 3-hydroxy-3-methylglutaryl (HMG-CoA)

reductase inhibitors, LDL receptor up-regulators, bile acid reabsorption inhibitors, cholesterol-7α-

hydroxylase (CYP7A1) activators, and plasma cholesteryl ester transporting protein (CETP) inhibitors.

This presentation will discuss our research progress in the field, and explore the underlying mechanisms

of some popular cholesterol-lowering nutraceuticals and functional foods.

Key words: Cholesterol, nutraceuticals, heart disease


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Study of process conditions for obtaining black soymilk

Thiana, ESTEVES

1; Ilana, FELBERG

2; Adelia, FARIA-MACHADO

2; Ronoel, GODOY

2; Manuela,

SANTIAGO2; Sidney, PACHECO

2; Veronica, CALADO

1; Mercedes, CARRÃO-PANIZZI

3

1 Escola de Química, Universidade Federal do Rio de Janeiro, Brazil

2 Embrapa Agroindústria de Alimentos, Brazil

3 Embrapa Trigo, Brazil

Black soybeans are known as an herbal and health-food ingredient for hundreds of years in the

Eastern Medicine mainly due of the bioactive compounds, especially anthocyanins. However, little

information is available about black soybean healthy food products possessing, like soymilk or

beverages. Preliminary studies showed that black soymilk made from milled soybean instead of

whole grain presented higher anthocyanins content and antioxidant activity. The objective of this

study was to identify the best conditions for black soymilk processing, considering cooking time and

process temperature, by using a 22

factorial design with three central points. Temperature ranged

from 80°C to 98°C and cooking time from 5 to 15 minutes. Anthocyanins (mg/100g) and isoflavones

(aglycon equivalent mg/100g) were determined by HPLC systems and antioxidant activities were

estimated using 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging and oxygen radical

absorbance capacity (ORAC). We concluded that time and temperature were significant (p ≤ 0.05)

for anthocyanins content and antioxidant activity. Isoflavone content ranged from 138.74 (98°C/15

min) to 148.68 (89°C/10 min) mg/100g dry wt; anthocyanins content ranged from 41.03 (80°C/15

min) to 49.97 (80°C/5 min) mg/100g dry wt.The 5min cooking time/80°C was the best black soymilk

processing condition considering all the parameters evaluated in this study.

Keywords: Black soymilk, process, antioxidant activity


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Influence of Maternal Nutritional Status on Pregnancy Outcomes

Parmeet Kaur1, Suneeta Mittal

2, Arun Kumar

3, Tulsi Adhikari

4, Neeta Kumar

5

1. Department of Dietetics, AIIMS, New Delhi.

2. Department of Obstetrics & Gynecology, FMRI, New Delhi

Background and Objective: Maternal nutritional status is recognized as a very important regulator of

foetal growth. In India, meagre information is available on nutritional intake of pregnant women

related to birth outcomes. Therefore, the present study was undertaken to study the nutritional status

during pregnancy and compare with the available recommended dietary allowances (RDAs) and

correlate with birth weight of the newborn Methods: Nutritional assessment of 240 pregnant women

attending the antenatal clinic of premier tertiary care hospitals of northern India was done.

Results: Mean energy and protein intake during the entire period of pregnancy were much lower than

the recommended dietary allowance (RDA). The energy and protein consumption deficit lowered

from 23.5% to 13.6% and 32% to 17.6%, from baseline towards the end of third trimester,

respectively. Dietary intake of Iron was found to be 53% lower at baseline and 49.4% lower than the

RDA at the end of third trimester. The mean weight gain during pregnancy was also found to be

significantly low and correlated with caloric intake.

Conclusion: There was significant correlations between the maternal nutrient intake and birth weight

(p=0.01).Energy and protein intake was found to be positively correlated with birth weight. The

present study reflects the relationship between nutritional intake and weight of the newborn.

Therefore, more emphasis is required to bridge the evident food gap through nutrition education

programs.

Keywords: Dietary intake, pregnancy, birth outcomes


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Efficacy of fish oil-derived n-3 polyunsaturated fatty acids on inflammatory and

oxidative stress markers in a randomized controlled trial of type 2 diabetes mellitus

patients with abdominal obesity

Feng, Wang; Yanyan, Wang; Hui, Xia; Xian, Yang; Guiju, Sun

*

Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, and Department of Nutrition and Food Hygiene, School of Public Health, Southeast University, Nanjing

210009, China

Effects of fish oil supplements on inflammation and oxidative stress are insufficiently clarified in type

2 diabetic patients with abdominal obesity. This study aimed to determine the effects of n-3

polyunsaturated fatty acids (PUFA)-containing fish oil on the serum levels of inflammatory and

oxidative stress markers in type 2 diabetic patients with abdominal obesity. In a double-blind,

placebo controlled trial, 100 type 2 diabetic patients with abdominal obesity were randomized into

two groups including 4 g/day of fish oil (2.5 g n-3 PUFA) or placebo (corn oil) for 6 months. At the

beginning of the study and post intervention, fasting blood samples were taken and serum

concentrations of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6),

superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and 8- iso-

prostaglandin F2α were assessed and compared. Fish oil supplementation did not significantly affect

serum CRP, TNF-α, IL-6, SOD, GSH-Px and 8- iso- prostaglandin F2α as compared with control

subjects. However, there was a trend for MDA to decrease after fish oil supplementation (P=0.083).

In conclusion, this 6 months randomized, double-blind placebo-controlled intervention trial showed

2.5 g/day n-3 PUFA had neutral effects on inflammation and oxidative stress in type 2 diabetic

patients with abdominal obesity. This trial was registered at chictr.org as ChiCTR-TRC-14005084.

Keywords: n-3 polyunsaturated fatty acids; Type 2 diabetes mellitus; Inflammation; Oxidative stress


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An Empirical Analysis Of The Death Of Children Under Five Years In Rwanda

Fidence Munyamahoro

Research Scholar, Department of Actuarial Sciences, University of Lausanne, Switzerland. Malnutrition continues to be a primary cause of ill health and mortality among children in developing

countries. It is a major public health problem and accounts for about half of all children deaths

worldwide (UNICEF2004). The aim of this paper is to identify factors which cause the death of

children under five years and the prevalence of them. The study applied quantitative method in which

3015 married women that have children less than five years in eastern province in Rwanda over the

period 2010 to 2011. The questionnaire was scientifically developed and used to collect the data.

The present study was undertaken among 3015 married women who have children less than five

years in rural area and urban area. The analysis in this paper was done with the help of simple

percentages and survival. The results indicate that 44.4% of the children less than five years die

because of malnutrition, 11.8% of the children less than five years die because of Diarrhoea, 17.1%

of the children less than five years die because of poisoning. . It is very important to encourage the

health workers to teach the mother to take in healthy food and also show them the wide varieties.

Key words: nutritious diet for child, socio economic and demographic characteristics of women who

have children less than five years


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Antineoplastic activity of the essential oils from Chrysanthemum morifolium Ramat

Guiju Sun, Zhixiu Song, Feifei Zhang, Ligang Yang

Key Laboratory of Environmental Medicine and Engineering of Ministry of Education, and

Department of Nutrition and Food Hygiene, School of Public Health, Southeast University, Nanjing

210009, China

The essential oils of Chrysanthemum morifolium Ramat were extracted, and the antineoplastic

activity was studied. In vitro, cell proliferation inhibition action on human liver cancer HepG-2 of

essential oils extracted from Chrysanthemum was examined by MTT method and cell apoptosis was

observed using flow cytometry. In vivo, we used S-180 tumor-bearing mice to research anti-tumor

effect of the oils. The oils showed significant antitumor activity towards HepG-2 cells, with IC50 values

1.4232µl/ml, and increased the apoptotic rates in a dose dependent manner. In addition, the oils

inhibited tumor growth of S-180 tumor-bearing and the tumor suppressor rate of high dose reached

54.94%. Giving the oils improved the thymus/weight index of tumor-bearing mice which means it

increased immune function. All these findings suggest that the essential oil from Chrysanthemum

morifolium Ramat has antineoplastic activity. It is a candidate for exploring new anti-cancer agents.

[This study was funded by Jiangsu Province Science and Technology Support Program (No.

BE2006318)].

Keywords: ·Antineoplastic activity; Chrysanthemum morifolium Ramat; Essential oil


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Hypolipidemic effects of n-3 PUFA on Type 2 Diabetes with hyperlipidemia in

postmenopausal women

Hui, Xia; Yanyan, Wang; Feng, Wang; Xian, Yang; Guiju, Sun*

Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, and Department of Nutrition and Food Hygiene, School of Public Health, Southeast University, Nanjing 210009, China

Objective The study aims to investigate the hypolipidemic effects of n-3 PUFA on metabolic

variables in postmenopausal women with type 2 diabetes. Method Physical examination and blood

test were administered in 31 outpatients with type 2 diabetes from Diabetes Specialty Clinic in

Guanlin Hospital, and dietary intakes were measured by food frequency questionnaire. The

intervention study was a double-blinded randomized placebo-controlled trial, 31 postmenopausal

women with type 2 diabetes were included in the study based on inclusion criteria and exclusion

criteria, and assigned to two groups of an intervention group or a control group. The participants

received 4 fish oil capsules/d (620mg total n-3 PUFA/capsule), or 4 corn oil capsules/d for a period of

6 months. Physical examination and biochemical detection were administered in 3-month

intervention period and 6-month intervention period respectively. Result There were no significant

difference in blood pressure, BMI (Body Mass Index), WHR (Waist Hip Rate), and indexes of glucose

metabolism between two groups. After 3-month intervention, n-3 PUFA decreased TG level by 33.4%

(P<0.05) and increased HDL (High Density Lipoprotein) level by 19.0% (P<0.05). After 6-month

intervention, n-3 PUFA decreased TG (triglyceride) level by 35.2% (P=0.110), n-3 PUFA increased

HDL level by 20.9% (P=0.142). Conclusion N-3 PUFA decreased TG level in postmenopausal

women with T2DM and hypolipidemic, and increased HDL level. Effects of n-3 PUFA on TG and HDL

showed time-dependent.

Keyword: n-3 PUFA; type 2 diabetes mellitus; anti-hyperlipidemia


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Effect of partially hydrolyzed guar gum (PHGG) on postprandial hyperglycemia, hyperlipidemia and incretins metabolic hormones in healthy and glucose intolerant

subjects

Mahendra P. Kapoor*, Noriyuki Ishihara, Tsutomu Okubo

Nutrition Division, Taiyo Kagaku Co. Ltd., Yokkaichi, Mie Japan.

Dietary fibers intakes have consistently been associated with reduced risk for the diabetes and

metabolic syndrome hazards. This 12 months pilot study of soluble dietary fiber PHGG, evaluated 12

subjects free of diabetes mellitus with fasting plasma glucose of 124 mg/dl and LDL 157 mg/dl.

Participants were characterized as NGT (n=6), IGT (n=4), and PDM (n=2) subjects according to their

postprandial plasma glucose levels based on cookie test (OGTT test). Administration of 6g/ PHGG

with each meal (3 times/day) for 12 months significantly lowered the postprandial plasma glucose

(p=0.013). A reduction in both fasting and postprandial insulin and triglycerides levels, and

triglyceride/apolipoprotein-48 ratio could be observed. LDL levels were lowered, while significant

increase in HDL levels were registered (p=0.05), hence resultant decreased in their ratio (p<0.01)

was notices. In presence of weight loss, the HbA1c, WC, and BMI were reduced compared with

baseline (p<0.05). PHGG did not significantly affect an increase of fasting GLP-1, whereas fasting

GIP was significantly reduced along with fasting glucagon levels (p<0.05). Plasma leptin and hs-CRP

concentrations were significantly reduced with PHGG supplementation (p<0.05). In fact, 3 out of 6

IGT and/or PDM subjects turned out to be NGT subjects even after 3 months of PHGG

supplementation. Therefore, inclusion of PHGG in diets could improve metabolic health profiles by

affecting circulating metabolites that are responsible for postprandial glycaemia, hyperinsulinemia

and hyperlipidemia factors.

Keywords: PHGG, hyperlipidemia, postprandial glycaemia, etc.


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Development of a post-fermented Tea and its Physiological Effects

Kieko Saito1, 2

, , Mitsuaki Sano2 and Yoriyuki Nakamura

2

1 School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka City, 422-8526, Japan,

2Tea Science Center, University of Shizuoka, Shizuoka City, 422-8526, Japan

We developed post-fermented tea to prevent a lifestyle-related disease and analyzed the taste, and

the radical-scavenging activity and anti-obesity effect of the post-fermented tea considered as

physiological functions. Traditional fermented foods are becoming more popular in Japan, especially

as the physiological functions of lactic acid bacteria is so popular worldwide. Pu-erh tea is a post-

fermented tea, which is fermented with microorganisms. However, since caffeine, one of the main

components of tea, results in unacceptable physiological effect, we used low caffeine tea to develop

post-fermented tea. To decrease the caffeine concentration, we treated freshly picked tea leaves with

a low caffeine processing machine and fermented the leaves with lactic acid bacteria (Lactobacillus

plantarum) for several weeks before drying them. The flavor of the post-fermented tea was tested

using a taste sensing system, resulting in a decrease in astringency and no change of bitterness. As

for physiological effects, the extract of the post-fermented tea exhibited an anti-oxidative property for

several weeks after the fermentation and inhibited cell differentiation in the adipocytes. In this study,

we developed post-fermented tea with a low concentration of caffeine, which was a novel tea product

showing anti-oxidative activity and preadipocyte differentiation-inhibitory activity and may prove to be

both a functional food and a palatable one.

Keywords: post-fermented tea, anti-oxidative activity, anti-obesity


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“Pan de muerto”, Mexican traditional bread: Understanding its characteristics and

staling by relating its fat content to starch retrogradation

Anne MATIGNON, Alberto TECANTE

Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma

de México, Ciudad Universitaria, México, D.F. 04510, México

“Pan de muerto”, a traditional Mexican bread, is composed of a high fat/starch ratio. It is a popular

belief that its fat content ensures its long-term preservation. The objective of this work was to study

the functionality of fat on the bread evolution with time.

After characterization of the original bread, fat functionality was studied. The formulation, fat type and

presence, and the storage, moisture and temperature conditions, were modified. The evolution of the

structures of bread over eight days was determined at different scale by different methods: 1)

macrostructure, mechanical properties, 2) microstructure, confocal microscopy and thermal

properties, 3) molecular, crystalline properties.

“Pan de muerto” structure was shown to be hybrid between bread and pastry products. The breads

macrostructure highlighted staling behavior not dependent on moisture content. The storage

conditions impacted 1) butter crystallization, but not that of lard despite its well-known polymorphism,

2) starch retrogradation rate but not its nature. The fat conditions allowed highlighting a new relation

between fat composition and thermal properties of retrograded starch, B-type crystals; in fat-

containing products they needed more energy to be melted. The bread microstructure highlighted

different localization and distribution of fat, starch and proteins. The differents level of organization

together with specific interactions between lard and bread matrix or starch components could explain

the behavior highlighted. Both aspects continue to be currently studied.

Keywords: Cereal products, Staling, Starch retrogradation


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The micro-nutrient Composition of Chloroplasts from Range of Green Leafy Tissues

Mohamed A. Gedi1’3, Felius Yuseli2, Rhianna Briars1, David A. Gray1

1Division of Food Science, School of Bioscience, University of Nottingham, Sutton Bonington

Campus, LE12 5RD, United Kingdom 2Division of Nutritional Sciences, School of Bioscience, University of Nottingham, Malaysia Campus,

Jalan Broga, 43500, Semenyih Salangor Darul Ehsan, Malaysia 3 Faculty of agriculture, Somali National University, Columbia Road, Hamarweine, Mogadishu,

Somalia

Selected green leafy tissues (Spinach; Spinacia oleracea, spring greens; Brassica oleracea var

acephala, nettles; Urtica dioica and grass; Paspalum notatum) and their chloroplast rich materials

(CRM) were analysed and compared for their composition of selected vitamins and minerals by high-

performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry

(ICP-MS). The results indicated that CRM and the total leafy tissues were rich in vitamins E and C,

expressed as α-tocopherol and ascorbic acid, respectively, as well as pro-vitamin A (β-carotene).

They were also moderately rich in some major (Mg, P, K, Ca) and trace (Fe, Mn, Zn and Cu)

minerals. Alpha-tocopherol and β-carotene were significantly (p=0.05) higher in CRM, whereas

ascorbic acid was significantly (p=0.05) more concentrated in the leafy matter. Decreased trend of

major minerals and increased pattern of iron in CRM compared to total leaf tissues was also

observed. Micro-nutrient deficiency, vitamin A and iron in particular, remain a significant global health

issue.

Keywords: Chloroplasts; micronutrients, green leaves.

http://en.wikipedia.org/wiki/Brassica_oleracea


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Increasing Broiler Carcass Yield by Protein and Methionine Supplementation

Abbas Mohammadpour1, Aliasghar Saki, Taghi Akhzar

1Ph. D. Student of Department of Agriculture Science, Shiraz University, Shiraz, Iran

The experiment was conducted with two levels of protein and three levels of methionine. Carcass

yield and breast meat were significantly (p < 0.05) increased by 23% Crude Protein (CP) compared

with 20%. Abdominal fat was significantly decreased by 23 and 20% CP diet. In the interaction of

20% CP and high level of methionine (0.46%) on carcass yield, a significant increase was found

compared with 18% CP and both 0.38% and 0.42% of methionine levels. A similar effect was found

on breast meat yield compared with interaction of both of 0.5% and 0.55% of methionine and 20%

CP at 21 days, as well as at 42 days of age with 0.38% methionine and 18% CP. No significant

differences were found for protein, methionine or their interaction on Feed Conversion Ration (FCR),

with the exception of an interaction of 20% CP and 0.46% methionine which significantly improved

the FCR compared with 18% CP and 0.38% level of Methionine. Finally, the results of this study,

have suggested that methionine/CP ratio could be adjusted at 0.024% in starter and 0.023% for

grower diets. Decreased CP in the diet, increased breast meat yield and reduction of abdominal fat

were achieved by these ratios of methionine/protein.

Key words: Protein, Methionine, Meat.


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Effects of atmospheric radio-frequency (RF) plasma on mold spoilage, color, nutritional value, and mineral content of the whole grain brown rice

Narumol Matan1*

and Mudtorlep Nisoa2

1Food Technology, School of Agricultural Technology, Walailak University, Nakhon Si Thammarat,

80161, Thailand

2School of Science, Walailak University, Nakhon Si Thammarat, 80161, Thailand

222 Thasala district, Nakhon Si Thammarat, 80160, Thailand

Whole grain brown rice (WGBR) has been consumed as food and medicine for hundreds of years.

WGBR are excellent sources of nutritious foods. The long term storage of WGBR is very significant.

The objective of this study was to investigate the effectiveness of atmospheric RF plasma (10W-

40W) on mold spoilage in WGBR. Studies of the changes in the color, nutritional value, and mineral

content of the WGBR before and after plasma treatment were also analyzed. The results showed

that RF plasma at 40W was found to be the most effective against mold spoilage on WGBR for up to

12 weeks; without the plasma treatment, mold covered the WGBR in only 4 weeks. After plasma

treatment, the a* and b* values of WGBR were decreased but the L* value was not affected. In

addition, after 12 weeks of storage, WGBR with plasma treatment was found to increase the L*

value, while the L* values of control were decreased. The RF plasma had no effect on the crude

protein, and elemental composition (P, K, Mg, Ca, Zn, Na, Mn, and Fe) of WGBR. Therefore, an

atmospheric RF plasma can be used for the preventing of WGBR from mold growth during storage

retained its fresh nutritional value and mineral content.

Keywords: Whole grain brown rice; plasma; nutritional value


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Evaluation of Potentials of some selected seeds’ flours as partial Substitute for Wheat in Cookies production

1 Omobolanle Olorode, 2Michael Idowu and 3Adefunke Bamgbose

1,3Department of Food Technology, Moshood Abiola Polytechnic, P.M.B. 2210, Abeokuta, Ogun

State, Nigeria 2Department of Food Science and Technology, Federal University of Agriculture, P.M.B. 2240,

Abeokuta, Ogun State, Nigeria

Increase in the consumption of fruit pulp usually result into seeds referred to as waste meanwhile

these seeds could be of value. Main objective of this study was to determine qualities of cookies

from wheat partially substituted with some selected seeds’ flours. Pawpaw, benoil, golden melon and

water melon seeds’ flours were substituted with wheat flour at ratios 50:50. Functional properties of

the flour blends were determined. Cookies were produced from the blends using standard method.

Nutrient composition, physical and sensory properties of the resulting cookies were determined. All

these results were subjected to statistical analysis (SPSS 17.0) and means separated. Incorporation

of different seeds’ flours resulted into high oil absorption capacity and least gelation capacity but

there was no significant (p>0.05) difference in bulk density. Incorporation of each seeds’ flours into

wheat flour for cookie production improved its fibre, ash, protein and virtually all the minerals

examined. The increase was mostly significant in cookies made with goldenmelon and watermelon

flours incorporation. Phytochemical such as flavonoid, alkaloid and tannin were high in cookies from

the blends which show that incorporation of these seeds’ flours in cookie making could help prevent

some diseases. Cookies made with watermelon and golden melon was accepted next to cookies

from wheat flour whereas cookie from pawpaw was least accepted. This study confirmed that great

potential exist for these seeds.

Keywords: Cookies; seed’s flour; Functional properties


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Effect of a beverage made of andean grains, quinoa, kaniwa and kiwicha in the

metabolic control of Obese Patients with Diabetes

Hugo Arbanil(3,d)

, Emily M. Toscano(1,a)

, Maria de la Luz Miranda(5,d)

, Miguel A. Inocente(1,b)

, M.L.

Guevara-Fujita(2,d)

, Ricardo Fujita (2,d)

, Benjamin Castaneda(1,d)

, Vera Novak(4,d)

, D.Patricia Garrido(1,c)

1Research Center in Traditional Medicine and Pharmacology (RCTM),

2Research Center of Genetics

(RCG) School of Medicine Universidad San Martín de Porras Lima-Peru 3Dos de Mayo Hospital

(DMH) 4Beth Israel Deaconess Medical Center-Harvard Medical School-Harvard University,

University of Guadalajara-Mexico(5).

(a) Bachelor in Biological Sciences, (b) Chemist, (c) Principal Investigator, (d) Co-Investigator

Evaluation of levels of LDL (LowdensityLipoprotein) and HDL (High DensityLipoprotein), HbA1c,

ALT-TGP (alaninoaminotransferasa), AST-TGO (aspartateaminotransferase), and how they have

changed in obese patients with type 2 Diabetes (T2DM) after the consumption of the beverage made

of Andean grains, quinoa, kañiwa y kiwicha (QKK) during eight weeks. Changes have been

compared with the placebo group during the same period of time. This clinical trial recruit 123

patient, 61 were treated with the beverage and 62 with placebo. All participants, treatment/placebo

were matched for age, sex, weight BMI (bodymassindex) > 30 and clinical treatment scheme. The

beverage used were previously tested for safety. In the treatment group levels of initial LDL

were114.8 mg/dL (S.D 37.3), p=0.666 and after eight weeks 102.3 mg/dL (S.D. 19.4) p= 0.628. In

control group LDL level initial 118 mg/dL final 105 mg/dL. In the treatment group levels of initial HDL

were 47.4 mg/dL (S.D. 16.2), final 49.2 mg dL (S.D 9.1, p= 0.042. HbA1c (glycated haemoglobin) in

treatment group initial 7.9% and final 7.3% (p= 0.62). In placebo group HbA1c was 8.2% and final

7.3%.The results suggest that the beverage has no effect between treatment and control group of

patients. However, appears to have some effect on cholesterol fractions in the treatment group. The

HbA1c shows a slightly change in the treatment group as well. We suggest further research using

this beverage and increasing patients sample size and timeframe for the intake beverage in the

patients with obesity and diabetes.


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Association of body composition and sleep behaviours in young Saudi females: A cross sectional survey.

Syeda Fatima Bushra

1; Shahida Banu Shamshuddeen

1; Eyad Alshammari

2; Epuru Suneetha

2;

Rafia Bano 2

1Lecturer, Dept. of Clinical Nutrition, University of Hail, Saudi Arabia

2Asst. Prof, Dept. of Clinical Nutrition, University of Hail, Saudi Arabia

Objective

To cross-sectionally assess potential associations between sleep behaviours and body composition

in a sample of young Saudi females

Methods

Participants (n=422, females , age 19 to 25 yrs) were studied for sleep patterns for one week with

the help of sleep diaries and week day sleep debt was calculated along with weekend sleep coping

practices. Participants were grouped into one of three groups (normal sleep for week days and

weekends (7- 9 hrs) (NS); sleep debt week days (< 7 hrs] and sleep coping on weekends (> 9 hrs)

(SDW with coping weekend); and sleep debt week days and weekends (< 7 hrs] (SD). Body

composition was measured using Bioelectric impedance analysis (BioSpace, Inbody 720).

Results

Around 16 % of study population was into SD group and another 36 % were into SDW with coping

weekend group. As compared to normal sleep group both i.e., SDW with coping weekend and SD

groups were having significantly higher means and higher odds for BMI, Neck Circumference,

Visceral Fat, Waist circumference and waist Hip Ratio except for percent body fat.

Conclusion

Young females with abnormal sleep timings and sleep patterns may be at high risk for obesity. Sleep

debt during week days with coping strategy on weekend may not be protective over long term effects

of sleep debt on metabolic disruption.


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Simultaneous Determination of Six Kinds of Nutritional & Functional Ingredients in Fresh Citrus Fruit by RP-HPLC-DAD Wavelength Switching Method

Weisheng, Xu; Zhu, Wang; Yuexin, Yang.

National Institute for Nutrition and Health, Chinese Center for Disease Control And

Prevention,China. Objective: The citrus fruit have gained wide attention for their nutritional value and health effects.

They are rich in a variety of natural nutritional and functional ingredients, and its effects on

preventing cancer, cardiovascular diseases and diabetes have been verified. A novel quantitative

method using RP-HPLC-DAD wavelength switching was developed for simultaneous determination

of four kinds of polyphenols (Hesperidin, Naringin, Naringenin, Hesperetinl) and two kinds of

limonoids (Limonin, Nomilin) in fresh citrus fruit.

Materials & Methods : The separation was carried out on an Agilent Eclipse XDB C18 column

(4.6mm×250mm, 5m) with temperature at 30°C. The 0.1% phosphoric acid (A) and acetonitrile (B)

as the mobile phase with linear gradient elution was set to a flow rate of 1.0ml/min. The gradient

program was as flows: 23%B isocratic (0~5min); 23%~55%B linear (5~15min), the detection

wavelengths were 270nm (0~5min) and 210nm (5~15min), respectively.

Main Findings: The developed calibration curves displayed good linearity over a concentration

range of

2~250mg/ml with all correlation coefficients higher than 0.9997. The recovery rate range was

94.6%~111.9%, with RSDs no more than 2%. The limits of quantification (LOQ) and detection (LOD)

were 0.40ng~4.77ng and 0.12ng~1.43ng, respectively.

Conclusion & Recommondations: The method has been verified to be accurate and reliable,

and can successfully used for the determination of the six kinds of nutritional ingredients in

different fresh citrus fruit samples.

Keywords: Polyphenol; HPLC; Citrus


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Evaluation of Waist Circumference, Waist/Hip Ratio, Waist/Height Ratio and Human Body Shape Index (HBSI) of University Students.

Sultan Nazif1, Seray Kabaran

1

1Nutrition and Dietetics Department, Faculty of Health Sciences, Eastern Mediterranean University,

Famagusta, TRNC

The aim of this study was to asses waist circumferences, waist/hip ratios, waist/height ratios and

HBSI of university students. 393 students (32,6% men, 67,4% women) aged between 17-28 (mean

age 21.0±2.11) were included in the study. HBSI, waist/hip ratio and waist/height ratio were used to

evaluate the body shapes. A bioelectrical analysis device (Tanita MC-980MA) was used to assess

the body compozitions. Tape measure was used to measure waist and hip circumferences. HBSI is a

new index developed as an alternative to Body Mass Index (BMI) and used to evaluate abdominal fat

and disease risk. [HBSI = Waist circumference / (BMI2/3

* height1/2

)]. Mean body weight, height, BMI,

waist circumference, hip circumference, body fat percent, total body fat mass, trunk fat mass was

found to be 63.5±14.17 kg, 166.3±8.71 cm, 22.7±3.76 kg/m2, 79.0±11.12 cm, 98.9±8.03 cm,

%23.8±6.38, 16.0±8.67 kg, 7.87±5.11 kg, respectively. Out of 393 students, 10.4% was underweight,

67.7% was normal, 16.0% was overweight and 5.9% was obese according to BMI classification.

Mean HBSI, waist/height ratio and waist/hip ratio were calculated to be 0.0764±0.005 (men;

0.0774±0.004, women; 0.0759±0.005), 0.47±0.05 (men; 0.50±0.05, women; 0.46±0.05), 0.79±0.06

(men; 0.85±0.05, women; 0.77±0.06), respectively. A positive correlation was found between HBSI

and waist/height ratio (r=0.509, p<0.000), waist/hip ratio (r=0.687, p<0.000), waist circumference

(r=0.513, p<0.000), body weight (r=0.104, p<0.05), total body fat mass (r=0.142, p<0.005), ve trunk

fat mass (r=0.167, p<0.001). When compared according to waist circumference classification

(normal, at risk, at high risk), it was shown that students at high risk group had higher mean HBSI,

waist/height ratio, waist/hip ratio and BMI than the other groups (p<0.05). This newly developed

HBSI can be evaluated as a indicator of increased abdominal fat of university students.

Key words: Waist circumference, Waist/hip, Waist/heigh, HBSI


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Body Composition of Soccer Referees in the Turkish Republic of Northern Cyprus

Fatma Hülyam, Eren1; Kamil, Dağcılar

1; Merve, Yanık

1; Özge, Yücel

1; Seher, Gültekin

1;

1 Eastern Mediterranean University, Faculty of Health Sciences, Nutrition and Dietetics Department,

Famagusta, North Cyprus.

The aim of this study was to examine the anthropometric characteristics of elite Turkish Cypriot

soccer referees. A portable bioelectrical impedance analyser TANITA BC 418-MA was used to

estimate the percentage of body fat (%BF). 71 male referees who belong to the Cyprus Turkish

Football Association (CTFA) participated in the first analysis in December 2014. Mean values for

age, weight, height, waist circumference, body mass index (BMI) and %BF were recorded as

28.7±7.5 years, 75.3±10 kg, 177.69±7.8 cm, 85.7±7.3 cm, 23.8±2.6 kg/m² and 17.6±9.0%

respectively. In this analysis referees were classified by division and role: 20 elite league referees,

12 elite league assistant referees and 39 classman referees. Analysing by division and role, there

were differences observed among the groups in age, weight, waist circumference and BMI

measurements but none of these results were statistically significant at p<0.05 level. Age, weight,

height, waist circumference and BMI mean values of referee groups were as follow: Elite league

referees: 33.15 years, 76.7kg, 178.7 cm, 88cm and 24 kg/m², elite league assistant referees 33.16

years, 79kg, 178 cm, 86.5cm and 24.7 kg/m² and classman referees 25.15 years, 73.5kg, 177cm,

84cm and 23 kg/m². Classman referees were the youngest age group, had the lowest weight and

waist circumference. There was no significant %BF difference observed among the three referee

groups. The overall %BF was refered to as healthy (elite referees 17.1%, elite league assistant

referees 17.5% and classman referees 17.8% BF). When compared, analysing by age groups, no

significant differences in any variable was found. To the best of our knowledge this is

the first study to analyse the the body composition of the Turkish Cypriot football league referees. As

a national record this study will be a good example for future studies. It can be suggested that

evaluation of body composition should be included in the CTFA fitness test for referees.

Keyords: Soccer, Referees, Body composition, Anthropometric

http://scholar.google.com.tr/scholar?q=Eastern+Mediterranean+University,+Nutrition+and+Dietetics+Department,&hl=tr&as_sdt=0&as_vis=1&oi=scholart&sa=X&ved=0ahUKEwjDlZasmMXMAhWGuBoKHdUcD9UQgQMIGDAA

http://scholar.google.com.tr/scholar?q=Eastern+Mediterranean+University,+Nutrition+and+Dietetics+Department,&hl=tr&as_sdt=0&as_vis=1&oi=scholart&sa=X&ved=0ahUKEwjDlZasmMXMAhWGuBoKHdUcD9UQgQMIGDAA


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The Effects of Ginseng Extracts on Perceived Stress, Blood Pressure, Bi-spectral

index and Pulse Oxygen Saturation in Normotensive healthy adults after exposure to white noise stressor.

Ik-Lyul Bae1, Wonjong Kim

2, Joomi Ahn

3, Mi kyoung Lee

4, Myung-Haeng Hur

4

1Daejeon Institute of Science and Technology, Department of Nursing, Daejeon, Korea

2Gimcheon University, Department of Nursing, Kyungbook, Korea

3Woosong University, Department of Nursing, Daejeon, Korea

4Eulji University, College of Nursing, Daejeon, Korea

Purposes This study was a was a randomized controlled trial to determine the effects of stress,

blood pressure, Bi-spectral index(BIS Index), pulse oxygen saturation(SPO2)of the ginseng extract

for a normotensive healthy adult. Methods The experimental treatment was to inhale ginseng

extracts, to be performed in the laboratory. Subjects were healthy normotensive adults, were

enrolled by recruit and were consented in participating study. The subject’s age is 25.0 years, and

their height, weight, blood pressure were 165.8cm, 59.0kg, 116.3 / 67.8mmHg. Before the treatment,

we measured the baseline data (pretest), and after hearing white noise as a loading the stressor, we

measured the stress loading data (TLS). After loading the stressor, we measured the initial data(T0)

before experimental treatment and then we gave ginseng extracts inhalation to the experimental

group, measured the posttest data (T5, T10, T15, T20, T25, T30) every 5 minutes during 30 minutes.

Data were analyzed by t-test, repeated measures ANOVA. Results General characteristics of

subjects showed no difference between the two groups. After experimental treatment, the perceived

stress, blood pressure, SPO2 were not significantly different between experimental group and control

group. But the BIS index of experimental group was lower than the control group (F = 14.02, p

= .001). Conclusions Ginseng extract was effective to lower the BIS index although not mitigate

the perceived stress level.

Keywords: Ginseng, Stress, Bi-spectral Index Monitor, Blood pressure


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TIgE level and IgE-mediated allergy of 3-12 years urban children in China

Yichun Hu, Wang Rui, Xiaoguang Yang, Lichen Yang.

National Institute for Nutrition and Health, China CDC, China

Background: The prevalence of allergic diseases has risen at an alarming rate throughout the

world. This study was to learn about the total IgE level, IgE-mediated allergy condition among urban

children in China. Methods: All the samples were selected from the serum bank built by the national

nutrition and health survey of Chinese residents. TIgE were tested by ELISA, and the sIgE were

tested by immunoblotting. Results: 755 cases of 5190 samples were found higher than the normal

range, among which, 365 cases of serum were detected allergic to food and/or respiratory allergens,

and the rate of IgE-mediated allergic reaction was 7.03% (3.20% food allergens, 5.00% respiratory

allergens). Main food allergens were milk, egg white, beef and nuts, and the main respiratory

allergens included mould mixture, dog dander and dust mite. Conclusions: Normal range of serum

TIgE obtained in this study was helpful for detecting IgE-mediated allergy. And the prevalence of

allergy in normal Chinese children could be preliminary analyzed by detecting TIgE and sIgE.

Keywords: TIgE, IgE mediated, 3-12 years old


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Control of Salmonella Enteritidis on fresh beef cuts with soy edible coatings

containing thyme and oregano essential oils

Gökçe Polat Yemiş1,*

and Kezban Candoğan2

1Sakarya University, Faculty of Engineering, Department of Food Engineering, Sakarya, Turkey 2Ankara University, Faculty of Engineering, Department of Food Engineering, Ankara, Turkey

Meat and meat products can be contaminated during the slaughter process with a variety of

foodborne pathogenic bacteria that may cause serious foodborne illnesses and deaths. Bio-active

packaging systems have been considered as a promising technology that has a significant effect on

shelf life extension and safety of the product. In this context, antimicrobial edible films and coatings

have gained great interest in preservation of foods due to their advantages such as their

biodegradable and environmentally friendly nature, and protection after the package is opened. In

this study, inhibitory effects of soy protein edible coatings incorporated with 1, 2, or 3% of thyme or

oregano essential oils (EOs) were determined against S. Enteritidis on fresh beef during refrigerated

storage for 14 days. The soy protein coatings with oregano and thyme EOs exhibited similar

antimicrobial activity against the test bacterium. Increasing concentration of both EO in edible

coatings resulted in significantly higher antibacterial activity for S. Enteritidis. The coatings

incorporated with oregano and thyme EO at 3% level exhibited reductions of 1.13 and 1.44 log unit

at Day 7 and 1.86 and 1.97 log unit at Day 14, respectively, in S. Enteritidis counts as compared to

control. This study demonstrated that application of edible coatings containing oregano and thyme

EOs on fresh beef cuts could have a potential in controlling S. Enteritidis with acceptable sensory

characteristics.

Keywords: Edible coatings, oregano essential oil, thyme essential oil, antibacterial activity


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Haze Formation in Clarified Pomegranate Juice and Concentrate

Oktay Yemiş1, 2*

and Hakime Gül Arslantürk1

1Pamukkale University, Faculty of Engineering, Department of Food Engineering, Denizli, Turkey 2Sakarya University, Faculty of Engineering, Department of Food Engineering, Sakarya, Turkey

Pomegranate juice is one the most important popular fruit juices due to the nutritional and health

benefits. The most important industrial problem in clarified pomegranate juices and concentrates is

the haze formation which is a main quality indicator during storage. In this study, pomegranates

(Punica granatum L. Hicaznar) were processed into juice by cold clarification method (4°C) using just

gelatin as a finning agent. In addition, reconstituted pomegranate juices (RPJ-1 and RPJ-2) were

produced from commercial pomegranate juice concentrates (PJC-1 and PJC-2) obtained from two

different fruit juice company. After pasteurization (90°C, 3 min), all pomegranate juices and

concentrates were stored at +4°C, +10°C and +20°C, and the development of haze was monitored

during 6 months. Pasteurization resulted in a considerable haze formation in both clarified and non-

clarified pomegranate juice although there was no effect of pasteurization on the haze formation in

reconstituted pomegranate juices. It was observed that the development of haze in clarified

pomegranate juices stored at +4°C was higher than stored at +10°C and +20°C juices. A dramatic

increase in haze development for all pomegranate juices was shown in the first 30 days of storage. It

was found that the development of haze in concentrate samples increased with rising temperature in

contrast to juice samples.

Keywords: Pomegranate juice, concentrate, haze formation, storage


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Hypoglycemic, Hypolipidemic and Antioxidant Activities of Musa Paradisiaca,

Normalis (Plantain) Supplemented Diet on Alloxan Induced- Diabetic Albino Rats

*Friday O. Uhegbu, Chinedu Imo, Chibuzo H Onwuegbuchulam and Ahamefule S. Ezekwe

Department of Biochemistry, Faculty of Biological & Physical Sciences, Abia State University, PMB

2000, Uturu, Nigeria

The hypoglycemic, hypolipidemic and antioxidant activities of unripe Musa paradisiaca (plantain)

supplemented diet on alloxan induced – diabetic albino rats were investigated. Results show that

30% Musa paradisiaca supplemented diet reduced significantly (p≤ 0.05) serum blood glucose, total

serum cholesterol and lipid peroxidation levels in the test animals. Antioxidant enzymes; superoxide

dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and gluthatione-s-transferase (GST)

levels increased significantly (p≤ 0.05) in the test animals. Serum blood glucose reduced from

223.42 ± 3.65mg/dl to 98.54 ± 2.36mg/dl, while total serum cholesterol and lipid peroxidation levels

reduced from 149.97 ± 1.35mg/dl to 133.23 ± 0.61mg/dl, and from 8.96 ± 0.65mg/ml to 6.87±

0.86mg/ml respectively. Superoxide dismutase, catalase, reduced glutathione and gluthatione-s-

transferase increased significantly (p≤0.05) from 25.30 ± 3.28 U/L to 32.72 ± 3.68 U/L; 2.76 ± 0.05

U/L to 3.45 ± 0.11U/L and from 2.86 ± 0.41U/L to 3.86 ± 0.62 U/L respectively in the test animals.

These effects are dose dependent, as it increases as the plantain percentage in supplemented diet

increases. The elicitation of these biochemical effects by the Musa paradisiaca supplemented diet

could be a reflection of its hypoglycemic, antilipidemic and antioxidant properties and could be of

nutritional and clinical importance in the management of diabetes and cardiovascular disease and

justifies the claim of alternative medicine practitioners that plantain could be used in the

management of diabetes.

Keywords: Cardiovascular disease, Cholesterol, Lipid Peroxidation,


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Food and nutrition data in the Wolfram Language

Andrew Steinacher

Scientific Content, Wolfram Research Inc

Some of the most popular queries on the computational knowledge website WolframAlpha.com

involve food and nutrition. In response to user interest, Wolfram Research has updated the new

Wolfram Language to allow users to have easier programmatic access to this data through this

computational knowledge framework.

With these updates, complex queries on thousands of foods involving their nutrition, colors, shapes

and images can be performed with codes shorter than the length of a tweet. By using the powerful

natural language processing built into the language, many queries do not require any code at all.

Additionally, the results of such queries can be easily visualized with histograms, word clouds, image

collages and many more using thousands of built-in visualization and computational functions.

Finally, all of these features can be used to deploy complete applications in the Wolfram Cloud, such

as a visual meal nutrition tracker that can be accessed on a wide variety of platforms.

This talk will give an overview of the food and nutrition data available in the Wolfram Language,

showing examples of the freely available open programming cloud as well as some analysis of

popular food queries from thousands of users of WolframAlpha.com.

Keywords: computation, data, visualization, applications


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Soybean Meal Extracts as Alternative to Antibiotics Use in Enteric Infections Control

Jenifer Mota, Rodrigues1; Maria Eugênia Rabello, Duarte

2; Miguel Daniel, Noseda

2

1Bioprocess Engineering and Biotechnology Postgraduate Program and

2Biochemistry and Molecular

Biology Dept., Federal University of Paraná (UFPR), Curitiba-PR, Brazil Salmonellosis is a common foodborne infection, reaching almost 100 million cases per year around

the world. Antibiotics are used to control enteric infections, but their prolonged use leads to the

emergence of resistant strains. Therefore, researches for novel molecules capable of inhibiting

bacterial adhesion to the host tissue as alternatives to antibiotics become urgent for health, industry

and livestock sectors. The present study aimed to evaluate the capability of soybean meal extracts to

adhere enterobacteria. For this, a soybean meal suspension was subjected to thermochemical

treatment (120°C, pH 2.0). After neutralization and centrifugation, the supernatant obtained was

subjected to a second thermochemical process (FS-A fraction). FS-A was deproteinized with

trichloroacetic acid (FS-Tp), and then treated with ethanol (FS-Es, FS-Ep). All fractions were

submitted to chemical and spectroscopic analysis and were evaluated in vitro for their ability to

adhere to Salmonella. The results were analyzed following a bacterial growth equation. FS-Ep

fraction had high carbohydrate content (82.7%) and low protein content (4.7%). The remaining

fractions (FS-A, FS-Tp, FS-Es) had higher protein levels (40.1-86.8%) and lower carbohydrate

content (8.7-42.6%). All fractions showed galactose, arabinose and uronic acid as main

monosaccharides and ability to adhere to enterobacteria. These results indicate the biotechnological

potential of the treated soybean meal extracts in the prevention of enteric infections, as an

alternative to the use of antibiotics in livestock.

Keywords: Soybean Meal; Enteric Infections; Enterobacteria; Bacterial adhesion.


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The Use of Pure and Commercial Pepsins in the Obtainment of Antihypertensive

Whey Peptides

Luísa O. L. da Rosa1, Tatiana L. Azevedo

2, Alexsandro A. Santos

2, Marilia P. Stephan

2, Danillo M.

Gomes3, José E. Silva-Santos

3, Caroline Melliger-Silva

2, Lourdes M. C. Cabral

2.

1Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil

2Embrapa Agroindústria de Alimentos, Rio de Janeiro, Brasil

3Laboratório de Farmacologia Cardiovascular, Universidade Federal de Santa Catarina,

Florianópolis, Brasil The consumption of whey hydrolysates has become more popular with the discovery of its functional

properties, such as antihypertensive potential. However, its high cost of acquisition remains a barrier

for some consumers and small industries. The aim of the present study was to generate

antihypertensive whey peptides, through enzymatic hydrolysis, catalyzed by pure (PPA) and

commercial (PPC) pepsins, comparing both obtainment processes. PPA presented an enzymatic

activity of 11-fold higher, and a cost of acquisition of 1400-fold higher when compared to PPC. The

hydrolysis processes were performed for 3h-reaction and the enzyme concentrations applied were of

0.4% and 1.6% for PPA and PPC, respectively. HPLC analysis showed that β-lactoglobulin was

partially hydrolyzed in both treatments, while α-lactalbumin was completely hydrolyzed during the

first minute in PPA, and between 1 and 2h in PPC treatment. SDS-PAGE gels showed lactoferrin,

serum albumin and caseins being hydrolyzed before 1-min-reaction in both experiments. Biological

analysis showed a vascular relaxation of 77.1% and 66.9% in rats’ denuded aortic rings, when

applied 10 mg mL-1

of PPA and PPC hydrolysates, respectively, showing that peptides also act

through independent endothelium pathways, besides the well-known angiotensin-converting-enzyme

inhibition. The results showed that an increment of 4 times in the commercial pepsin concentration

was enough to generate a whey hydrolysate with high antihypertensive potential and lower cost of

acquisition.

Supported by FAPERJ and CAPES


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Proteome Approach to Characterize Proteins Induced in Strawberry Leaves after Salicylic Acid Treatment

Caroline Mellinger-Silva

1, Marilia P. Stephan

1, Marcos Fonseca

1, Ursla Fernando

2, Christof

Rampitsch2

1Embrapa Agroindustria de Alimentos, Rio de Janeiro, Brazil

2Cereal Research Center, Agriculture and Agri-Food Canada, Morden, Canada

Strawberry is a fruit of great importance throughout the world, and as it is susceptible to a large

variety of phytopathogenic organisms, there is a need to develop new strategies to extend the fruit

shelf-life and plant health to replace the use of traditional pesticides. The aim of this work was to use

a proteomics approach to characterize the proteins induced in strawberry leaves after the use of

salicylic acid (SA), a natural plant-defense elicitor. Strawberries were grown in the presence or

absence of SA. Central leaves from the plant were collected and freeze-dried when fruits were

mature. Leaf proteins were extracted using acetone/trichoracetic acid precipitation method, and

analysed using two-dimensional electrophoresis (2DE). A total of 424 spots were observed in the

gels, from which 95 spots (22.4%) changed significantly after SA treatment: 85 were up-regulated in

the treated condition, 6 in the non-treated and 4 were expressed in only one of the conditions.

Digested proteins were identified through tandem mass spectrometry and they were involved in

various cellular responses and metabolic processes, including stress and defense mechanisms. By

the use of proteomic tools, the results demonstrated that SA treatment was able to induce the

expression of strawberry leaf proteins and may be further considered as a candidate treatment for

replacing traditional pesticides, enhancing the fruit quality after all. Funded by FAPERJ.


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PAPERS


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A Proposal of Process Conditions for Obtaining Black Soymilk

Thiana, ESTEVES

1; Ilana, FELBERG

2; Adelia, FARIA-MACHADO

2; Ronoel, GODOY

2; Manuela,

SANTIAGO2; Sidney, PACHECO

2; Veronica, CALADO

1; Mercedes, C. CARRÃO-PANIZZI

3

1 Escola de Química, Universidade Federal do Rio de Janeiro, Brazil

2 Embrapa Agroindústria de Alimentos, Brazil

3 Embrapa Trigo, Brazil

Universidade Federal do Rio de Janeiro (UFRJ)

Escola de Química (EQ)

Abstract

Black soybeans are known as an herbal and health-food ingredient for hundreds of years in the

Eastern Medicine, mainly because of their bioactive compounds, especially anthocyanins. However,

little information is available about processing black soybean healthy food products, like soymilk or

soy beverages. Preliminary studies showed that black soymilk, made from milled soybean instead of

whole grain, presented higher anthocyanins content and antioxidant activity. The objective of this

study was to identify the best conditions for black soymilk processing, considering cooking time and

process temperature, by using a 22

factorial design with three central points. Temperature ranged

from 80°C to 98°C and cooking time from 5 to 15 minutes. Anthocyanins (mg/100g) and isoflavones

(aglycon equivalent mg/100g) were determined by HPLC systems and antioxidant activities were

estimated using 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical scavenging and oxygen radical

absorbance capacity (ORAC). We concluded that time and temperature were statistically significant

(p ≤ 0.05) for anthocyanins content and antioxidant activity. The 5 min cooking time at 80°C was the

best black soymilk processing condition considering all the parameters evaluated in this study.

Keywords: Black soybean, soymilk processing, antioxidant activity, anthocyanins, isoflavones

1 Introduction

Black soybeans have been used as a health food and herbal extract for hundreds of years in

Oriental medicine (XU and CHANG, 2008a). More recent studies attribute their beneficial health

effects to the presence of bioactive compounds (CHENG, LIN and LIU, 2011), especially

anthocyanins, present in the black seed coat (HA et al, 2009). Anthocyanins are flavonoids, red or

purple color, recognized as health-promoting functional ingredients due to their antioxidant capacity

(KOH, YOUN and KIM, 2011).

In general, heating processing promotes modifications in the total phenolic compounds,

flavonoids, anthocyanins and the antioxidant activity (XU and CHANG, 2008b). Some studies with

black soybean seeds and traditional eastern derivates, as natto and tofu, evaluated the processing

conditions and their influence on bioactive compounds profile and antioxidant activity (XU and

CHANG, 2008b; KIM et al, 2011). However, there is a lack of information about black soymilk and

beverages processing. To better preserve black soymilk’s bioactive compounds and antioxidant

activity, this study aimed at identifying the best conditions for black soymilk processing, considering

cooking time and process temperature, by using a 22 factorial design with three central points.


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2 Method and Materials

2.1 Black Soybeans Black soybean line (BRM 09-50995) was obtained from the Soybean Breeding Program for

Human Consumption of Brazilian Agricultural Research Corporation (EMBRAPA), harvested in

2014. This line has black seed coat and yellow cotyledon.

2.2 Experiment Design The combined effect of cooking time (X1) and temperature (X2) on the anthocyanins and

isoflavones contents and estimated antioxidant activities (DPPH and ORAC methods) of black

soymilk was evaluated by using a 22 factorial design with three central points, totaling seven

randomized experiments (E1= 80°C/5 min; E2= 80°C/15 min; E3= 98°C/5 min; E4= 98°C/15 min;

E5= 89°C/10 min; E6= 89°C/10 min; E7= 89°C/10 min).

Temperature (from 80°C to 98°C) and cooking time (from 5 to 15 minutes), as shown in Table

1, were selected based on literature (MORAES FILHO, 2014) and on previous studies for yellow

soybeans (FELBERG et al., 2009).

Table 1. Coded levels, temperature and cooking time for 22 factorial

design of the black soymilk processing conditions

Independent

Variables

Coded levels

-1 0 1

Time (min) 5 10 15

Temperature (°C) 80 89 98

2.3 Black Soymilk Processing

Selected seeds were washed, dried and grounded in a hammer mill (Perten Laboratory Mill

3100) to increase surface contact. Processing was carried out by cooking milled black soybean with

water (1:10; w:w), considering the four different treatments and three replicates at the central point.

After cooking, the slurry was homogenized in a Waring® Blender for 2 minutes and centrifuged in a

IEC Model K7165 centrifuge, containing a 150 micra nylon filter, at 4000 rpm for 5 min. The seven

black soymilk samples obtained were frozen (-18 °C), lyophilized and milled to evaluate the

anthocyanins and isoflavones content and estimate the antioxidant activity (DPPH and ORAC).

2.4 Methanolic Extract of Black Soymilk Samples

Methanolic extracts of black soymilk samples were obtained according to Pereira et al. (2014)

and used for anthocyanin content analysis and for estimating antioxidant capacity (DPPH and

ORAC). One gram of each lyophilized black soymilk sample was extracted twice with extraction

solution (methanol/water/hydrochloric acid 60:39:1) in a water bath (IKA HEIZBAD HB-250) at

50°C for 1 hour, homogenizing every 10 minutes by vortexing for 5 seconds. After extraction, the

mixture was centrifuged (SORVALL LEGEND Centrifuge XRT) at 12.000 RPM for 10 min at

25°C. The supernatant was completed to 50 mL (volumetric flask) with the extraction solution,

filtered through a PTFE membrane CHROMAFIL Xtra – 45/25, pore size: 0.45 M, filter-Ø: 25 mm

and stored in amber bottle at –18°C. Samples were taken in duplicate.


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2.5 HPLC Analysis of Anthocyanin Content

Chromatographic analysis was performed according to Santiago et al. (2010), using a Waters®

Alliance 2695 system equipped with a Waters® 2996 photodiode array detector. Anthocyanins were

separated on a Thermo® Scientific C18 BDS (100 mm × 4.6 mm; 2.4 μm) column, using a gradient

of acetonitrile and 5% aqueous formic acid as mobile phase at 1 mL/min. Column temperature was

set at 30°C. Chromatograms were processed at 520 nm, using Empower® software (Waters).

Anthocyanins were quantified by external standardization (SANTIAGO et al., 2010).

2.6 Antioxidant Activity

Antioxidant activity of black soymilk was estimated using Radical DPPH Scavenging Activity

assay and Oxygen Radical Absorbing Capacity Assay (ORAC). DPPH assay was run according to

the method described by Brand-Williams et al. (1955). Briefly, an aliquot (0.1 mL) of different

dilutions of the methanolic extracts was mixed with 3.9 mL of DPPH methanolic solution;

deactivation of free radicals was determined after 15 minutes of reaction by absorbance reading at

515 nm using a spectrophotometer. Antioxidant activity was expressed as the amount of extract

required to reduce 50% of the initial concentration of DPPH (EC50). The ORAC assay was

conducted according to Huang et al. (2002). Black soymilk extracts were diluted 7, 10, 13, 16 and 20

times in phosphate buffer pH 7.4. Initially 25 L of sample, standard or blank solution was pipetted

into a 96-well plate, added 150 L fluorescein (61.2 nM final concentration) and kept at 37°C for 30

min. Then, 25 L of AAPH (19.1 mM final concentration) were added to each well and the

fluorescence at 528 nm was monitored for 60 min at 37°C. Quercetin was used as a reference

antioxidant because of its greater chemical similarity to anthocyanins as compared to Trolox,

compound commonly used as reference antioxidant in this method (HUANG et al., 2002).

2.7 Extraction and HPLC Analysis of Isoflavone Content

Extractions and analysis of isoflavone aglycones (genistein, daidzein and glycitein) and

glycosides (genistin, daidzein and glycitin) from black soymilk were performed according to AOAC

(2005) method 2001.10. Isoflavones identification and quantification were performed by comparison

of the investigated peak retention time and UV spectrum with those of the respective standards.

Results of total isoflavones were expressed as aglycon equivalents.

2.8 Statistics

Experimental design and statistical analysis were carried out by STATISTICA software, version

12.0 to Windows (StatSoft®). Analysis of variance (ANOVA) and Fisher’s test (LSD) were used to

verify significant differences (p ≤ 0.05) in anthocyanins, isoflavones and antioxidant capacity assays

DPPH and ORAC means among samples.

3 Results

3.1 Anthocyanin Content

Black soymilk anthocyanins content from different processing conditions ranged (mean scores)

from 39.75 to 50.47 mg in 100g of lyophilized samples (Figure 1). Treatments E1 (80°C/5 min) and

E3 (98°C/5 min) showed the highest anthocyanins contents as 49.75 mg/100g and 50.47 mg/100g,

respectively, and no significant difference between them. Three anthocyanins were detected:

delphinidin-3-glucoside, petunidin-3-glucoside and cyanidin-3-glucoside; the last one is the major

anthocyanin in black soymilk.


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Figure 1. Anthocyanins contents (mean scores) for the seven black soymilk treatments (E1 = 80°C/5

min; E2 = 80°C/15 min; E3 = 98°C/5 min; E4 = 98°C/15 min; E5 = 89°C/10 min; E6 = 89°C/10

min; E7 = 89°C/10 min).

3.2 Isoflavone Content

Four isoflavones (daidzin, glycitin, genistin and daidzein) were identified in the black soymilk.

Quantification was based on aglycone equivalents. The total isoflavone content ranged (mean scores)

from 138.7 mg/100g (E4 = 98°C/15 min) to 148.7 mg/100g (E7 = 89°C/10 min), respectively

(Figure 2).

Figure 2. The total isoflavone contents (mean scores), aglycone equivalents, for the seven black

soymilk treatments (E1 = 80°C/5 min; E2 = 80°C/15 min; E3 = 98°C/5 min; E4 = 98°C/15 min; E5 =

89°C/10 min; E6 = 89°C/10 min; E7 = 89°C/10 min).

3.3 Antioxidant Activity

Antioxidant activity estimated by DPPH and ORAC assays is presented in Figure 3. The EC50

of DPPH scavenging activity ranged from 0.29 mg/L (E7 = 89°C/10 min) to 0.39 mg/L (E3 = 98°C/5

min). Once DPPH assay results were expressed as EC50, lower values mean higher antioxidant

activity. The ORAC values ranged from 0.31 mM (E7) to 0.46 mM (E1 = 80°C/5 min) quercetin

equivalent, and E1 showed the higher antioxidant activity.

0

10

20

30

40

50

E1 E2 E3 E4 E5 E6 E7

Anthocyanins (mg/100g)

130

135

140

145

150

E1 E2 E3 E4 E5 E6 E7

Isoflavones (mg/100g)


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Figure 3. Antioxidant activity estimated by DPPH and ORAC assays for the seven black soymilk

treatments (E1 = 80°C/5 min; E2 = 80°C/15 min; E3 = 98°C/5 min; E4 = 98°C/15 min; E5 =

89°C/10 min; E6 = 89°C/10 min; E7 = 89°C/10 min).

4 Conclusions

Considering the anthocyanins contents and ORAC assay results, the 5 min cooking time at 80°C

(E1) seemed to be the best black soymilk processing condition. Further research needs to be carried

out in order to evaluate pasteurization treatment and to estimate shelf life of the final product.

5 Acknowledgments

The authors would like to thank EMBRAPA, FAPERJ and CNPQ for the financial support.

6 Reference List

AOAC Official Method, 2001.10. 2005. Determination of isoflavones in soy and selected foods

containing soy. In: Official Methods of Analysis, 17th edn. Chapter 45.4.14. AOAC International,

Gaithersburg, MD, USA.

Brand-williams, W.; Cuvelier, M. E.; Berset, C. 1995. Use of a free radical method to evaluate

antioxidant activity. Food Science and Technology, v. 28, p. 25-30.

Cheng, K. C., Lin, J. T. & Liu, W. H. 2011. Extracts from fermented black soybean milk exhibit

antioxidant and cytotoxic activities. Food Technology and Biotechnology, 49, 111-117.

De Moraes Filho, M. L., Hirozawa, S. S., Prudencio, S. H., Ida, E. I., Garcia, S. 2014. Petit Suisse

from black soybean: bioactive compounds and antioxidant properties during development process.

Int J Food Sci Nutr, v. 65(4), 470-475.

Felberg, I., Antoniassi, R., Deliza, R., De Freitas, S. C., Modesta, R. C. 2009. Soy and Brazil nut

beverage: processing, composition, sensory, and color evaluation. Ciência e Tecnologia de

Alimentos, v. 29(3), 609-617.

Ha, T. J., Lee, J. H., Shin, S.-O., Shin, S.-H., Han, S.-I., Kim, H.-T., Ko, J.-M., Lee, M.-H. & Park,

K.-Y. 2009. Changes in anthocyanin and isoflavone concentrations in black seed-coated soybean at

different planting locations. Journal of Crop Science and Biotechnology, v. 12, 79-86.

0.000

0.100

0.200

0.300

0.400

0.500

E1 E2 E3 E4 E5 E6 E7

DPPH EC50 (mg/L)

ORAC

QUERCETIN

EQUIVALENT

(mM)


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Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., Prior, R. 2002. High-throughput assay of

oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled

with a microplate fluorescence reader in 96-well format. Journal of Agricultural and Food

Chemistry, v. 50, 4437– 4444.

Kim, H. G., Kim, G. W., Oh, H., Yoo, S. Y., Kim, Y. O. & Oh, M. S. 2011. Influence of roasting on

the antioxidant activity of small black soybean (Glycine max L. Merrill). LWT - Food Science and

Technology, v. 44, 992-998.

Koh, K., Youn, J. E. & Kim, H. S. 2014. Identification of anthocyanins in black soybean (Glycine

max (L.) Merr.) varieties. J Food Sci Technol, v. 51, 377-81.

Pereira, J. N.; Godoy, R. L. O.; Felberg, I.; Esteves, T. C. F.; Santiago, M. C. P. A.; Carrão-Panizzi,

M. C. 2014. Avaliação de metodologias de extração e caracterização do perfil de antocianinas em

soja preta por cromatografia líquida de alta eficiência (CLAE) e espectrometria de massas (MS).

XXIV Congresso Brasileiro de Ciência e Tecnologia de Alimentos. Aracajú-SE. Brazil.

Santiago, M. C. P. A. ; Gouvêa, A.C.M.S. ; Godoy, R. L. O. ; Oiano-Neto, J.; Pacheco, S. ; Rosa, J.

S. 2010. Adaptação de um método por cromatografia líquida de alta eficiência para análise de

antocianinas em suco de açaí (Euterpe oleraceae Mart.). Rio de Janeiro: Embrapa Agroindústria de

Alimentos (Comunicado técnico, 162. Biblioteca: CTAA (FL CTE 0162 UMT)).

Xu, B. & Chang, S. K. 2008a. Antioxidant capacity of seed coat, dehulled bean, and whole black

soybeans in relation to their distributions of total phenolics, phenolic acids, anthocyanins, and

isoflavones. J Agric Food Chem, 56, 8365-73.

Xu, B. & Chang, S. K. 2008b. Total phenolics, phenolic acids, isoflavones, and anthocyanins and

antioxidant properties of yellow and black soybeans as affected by thermal processing. J Agric Food

Chem, 56, 7165-75.


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AN EMPIRICAL ANALYSIS OF DEATH OF CHILDREN UNDER FIVE

YEARS IN RWANDA

Fidence MUNYAMAHORO

Research Scholar, Department of Actuarial Sciences, University of Lausanne, Switzerland.

Abstract

Malnutrition continues to be a primary cause of ill health and mortality among children in

developing countries. It is a major public health problem and accounts for about half of all children

deaths worldwide (UNICEF2004). The aim of this paper is to identify factors which cause the death

of children under five years and the prevalence of them. The study applied quantitative method in

which 3015 mothers that have children less than five years investigated. The analysis in this paper

was done with the help of simple percentages and survival analysis. The results indicate that 44.4%

of the children less than five years die because of malnutrition. This is much closed to the one of the

2010 Rwanda Demographic and Health Survey, 11.8% of the children less than five years die

because of Diarrhea, 17.1% of the children less than five years die because of poisoning. It is very

important to encourage the health workers to teach the mother to take in healthy food and also show

them the wide varieties.

Key words: Under-five mortality rate, nutritious diet for child, socio economic and demographic

characteristics of mother.

1. Introduction

Undernourishment affects physical appearance and energy levels, also directly affects many aspects

of the children’s mental functions, growth and development which have adverse effects on children’s

ability to learn and process information and grow into adults that are able to be productive and

contributing members of society. Undernourishment also impairs immune function leaving them

more susceptive to infection. Malnourished children, particularly those with severe acute

malnutrition, have a higher risk of death from other common childhood illness. Children with

infections are more susceptible to malnutrition and the cycle of poverty and malnutrition continues.

The best global indicator of children’s well-being is growth. Assessment of growth is the single

measurement that best defines the nutritional and health status of children, and provides an indirect

measurement of the quality of life of the entire population (Joshi HS et al 2011).


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Keeping an eye on child’s live condition is very important. Ensuring that a child’s nutrition is

incorporated with healthy nutritious diet, teaching them proper hygienic measures, letting them learn

the importance of exercises and having them visit their doctors and dentists will help them stay on

track with their health. Help your kids know the importance of being and staying healthy. Give them

a healthy body, healthy environment and a healthy family (Calk Arnold 2010). Malnutrition

continues to be a primary cause of ill health and mortality among children in developing countries. It

is a major public health problem and accounts for about half of all children deaths worldwide

(UNICEF2004). About 150 million children in developing countries are still malnourished and more

than half of underweight children live in South East Asia Region (UNICEF2004). Besides poverty

there are other factors that directly or indirectly affect the nutrition status of children. The main

studies showed that maternal education is considered as one of major element of an overall strategy

to address malnutrition (Christiaensen L 2001). According to the 2010 Rwanda Demographic and

Health Survey 44% of all children in Rwanda are chronically malnourished or stunted – shorter than

they should be for their age mostly due to insufficient food intake, recurrent illnesses, lack of

knowledge about proper infant and young child feeding, inadequate hygiene and sanitation, poor

primary health care, and household food insecurity.

2. Historical back ground

5.9 million Children under age five died in 2015, in average 16 000 children under age five died

every day.

The risk of a child dying before completing five years of age is still highest in the African Region

(81 per 1000 live births), about 7 times higher than that in the European Region (11 per 1000 live

births). Fortunately, under-five mortality rate has decreased by 53%, from an estimated rate of 91

deaths per 1000 live births in 1990 to 43 deaths per 1000 live births in 2015. ( WHO report) In

Rwanda, they made a valuable effort to reduce the childhood mortality from 152 per thousand lives

in 1990 up to 42 per 1000 lives in 2015.

2.1. Under-five mortality rate

Under-five mortality or child death also known as Child mortality can be defined as the death of

infants and children under the age of five or between the ages of one month to fifty nine months

depending on the definition. Child survival interventions are designed to address the most common

causes of child deaths that occur, which include pneumonia, malaria, diarrhea and neonatal

conditions. A child's death is emotionally and physically hard on the parents. The figure 1 shows the

Under-five mortality rate in Rwanda in the period of 1981-2015(WHO)

https://en.wikipedia.org/wiki/Pneumonia

https://en.wikipedia.org/wiki/Malaria

https://en.wikipedia.org/wiki/Diarrhea


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Figure1: The histogram of mortality rate, under 5 in Rwanda


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From the figure, the highest mortality rate appeared is 300 per 1000 in 1994 and 268 per 1000 in

1995 after that period the mortality rate reduced rapidly up to 42 per 1000 in 2015.

Like many countries of African region, the mortality rate under five in Rwanda is still high. The rate

in 2015 is 42 per 1000 lives; this rate is quite smaller than the rate of African region (81 per 1000)

but is not small enough compared to developed countries.

Figure2: Scatter plot of mortality rate

From the figure, the mortality rate decreased up to year of 1989 and then increased up to year of

1994 and after that period, the mortality rate decreased rapidly. The highest rate (three children

out of ten) appeared in 1994 because of genocide. It is very clear that the government of Rwanda

made an effort to reduce the death of children. Rwanda has almost 40 district hospitals and 400

health centers serving people, increasing these centers helped to reduce the death of children. And

some effort was put in the training of more community health workers (village health teams) to


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sensitize the community the following: to ensure that the baby is breathing, starting the newborn

on exclusive breastfeeding right away up to thousand days, washing hands before touching and

feeding the baby, and keeping the baby warm.

3. Data Analysis, Methods and Materials:

The study applied quantitative method in which 3015 mothers were sampled. The questionnaire was

scientifically developed and used to collect the data. The study was undertaken among 3015 mothers

who have children less than five years in rural and urban area. The analysis in this paper was done

with the help of simple percentages and cox-regression model.

3. 1. Findings

Table 2: Percentage distribution of died children according to age

Age of children (in month) Frequency Percent

0-12 1419 47.08

13-24 824 27.34

25-36 511 16.95

37-60 261 8.33

From the table, 47.08% of the children died before completing one year. This is obvious because a

child's risk of dying is highest in the neonatal period, the first 28 days of life. 27.34% died in a

period of between 13 and 24 months, 16.95% died in period of 25 and 36 months and 8.33% in

period of 37and 60 months.

Figure3: distribution of death respect to the age of child

Infant mortality or mortality during childhood is rapid decreasing of mortality during the first few

years of life. From the figure the number of death is too high in first 12 months.

0

500

1000

1500

[1-12] [13-24] [25-36] [37-60]

number of death


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Table 3: ages, education levels and marital status of mothers

Age of mother Frequency Percent

15 - 20 867 28.76

21 - 25 482 15.98

26 -30 483 16.02

31 -35 486 16.12

36 and above 697 23.12

Education level of mother

No Education 1477 48.99

Primary 1025 33.1

Secondary 513 17.01

Marital status of mother

Single mother 954 31.64

Married 724 24.01

Separated 1337 44.35

From this table age, education and marital status of mother have effect to the death of children. It is

seen that among mothers lose the children, 48.99% did not study, and 33.10% of them have primary

level only. The results show that a child from still married family has more chance of surviving than

others. 44.35% of died children were whom their mothers are separated.

To educate a woman is to educate a family (national), the education of mother has the great impact to

the life of children in family. From the table, most of died children was born to non-educated

mothers.

3. 2. Survival analysis.

Survival and hazard functions are common known in medical sciences especially in mortality

analysis. Let X be a continuous random variable with probability density function f(t) and

cumulative distribution function F(t) = Pr{X < t}, which is the probability that the event has occurred

by duration t. the survival function is the probability that the event of interest has not occurred by

duration t and given by: S(t) = Pr{T ≥ t} = 1 − F(t) = ∫

The hazard function is calculating the failure rate for ever smaller intervals of time and is defined by:

h(t) =

. The numerator of this expression is the conditional probability

that the event will occur in the interval [t, t+dt) given that it has not occurred before.

Hazard function also known as force of mortality) , the failure rate or hazard rate, h(t) is the ratio of

the probability density function f(t) to the survival function S(t), given by: h(t) =

The following figure shows the survival function at mean of covariates

http://mathworld.wolfram.com/ProbabilityDensityFunction.html

http://mathworld.wolfram.com/SurvivalFunction.html


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Figure7: survival functions at mean of covariates.

From this figure, it is also seen that before 10 months, all those kind of illness pass away so many

children especially diarrhea, malnutrition and malaria;between 10 and 20 months the most frequency

illnesses are malnutriton,poisoning and malaria;between 21 and 30 months, the most frequency

illnesses are malnutrition, poisoning and malaria;between 40 and 50 months poisoning and malaria

pass away the children and above 50 months many children died because of poisoning. Child

mortality rates related to respiratory and diarrheal diseases can be reduced by introducing

simple behavioral changes, such as hand washing with soap. This simple action can reduce the rate

of mortality from these diseases by almost 50 per cent.

4. Concluding remarks

The study shows that the more income range incease the less number of death happen, the house

holds use well/bore water get fewer illness and the good employement implies fewer kind of death.

The more nutritious diet for child intake the more the healthier they could be. It is very important to

encourage the child to take in healthy food and also show them the wide varieties of nutritious food

like green leafy vegetables, fruits, legumes and also to incorporate milk daily in their meals.

https://en.wikipedia.org/wiki/Diarrheal_diseases

https://en.wikipedia.org/wiki/Behaviour_change_(public_health)

https://en.wikipedia.org/wiki/Handwashing


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References

[1] WHO (1995c). WHO working group on infant growth. An evaluation of infant growth:

The use and interpretation of anthropometry in infants. Bulletin of the World Health

Organization, 73:165−74.

[2] Yip R, Scanlon K (1994). The burden of malnutrition: a population perspective. Journal

of Nutrition, 124:2043S−2046S.

[3] Finkelstin, Dianne M,ʽA Proportional Hasard Model for interval-censored Failure Time

Data’ in Biometrics

[4] UNICEF/WHO. Levels and trends in child mortality. Report 2013

[5] Kravdal . (2004) Child mortality in India: the community-level effect of education. Population

Studies [6] Allison, P.D.1995. Survival analysis: A practical Guide. Cary NC: SAS Institute.

[7]Sasieni, Peter, ʽsome new Estimators for Cox Regression’in The Annals of Statistical,

vol.21 Dec.1993

[8] Allison, P.D.1995. Survival analysis: A practical Guide. Cary NC: SAS Institute.

[9] Therneau, Terry M. And Granboch, Modeling Survival Data, Extending the Cox Model,

Springer, New York,2000

[10] Hill K. (1991). Approaches to the measurement of childhood mortality


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The Effects of Ginseng Extracts on Perceived Stress, Blood Pressure,

Bispectral index and Pulse Oxygen Saturation in Normotensive healthy

adults after exposure to white noise stressor.

Ik-Lyul Bae1, Wonjong Kim

2, Joomi Ahn

3, Mi kyoung Lee

4, Myung-Haeng Hur

4

1Daejeon Institute of Science and Technology, Department of Nursing, Daejeon, Korea

2Gimcheon University, Department of Nursing, Kyungbook, Korea

3Woosong University, Department of Nursing, Daejeon, Korea

4Eulji University, College of Nursing, Daejeon, Korea

Abstract

Purposes This study was a randomized controlled trial to determine the effects of stress, blood

pressure, Bispectral index(BIS Index), pulse oxygen saturation(SPO2)of the ginseng extract for a

normotensive healthy adult. Methods The experimental treatment was to inhale ginseng extracts, to

be performed in the laboratory. Subjects were healthy normotensive adults, were enrolled by recruit

and were consented in participating study. The subject’s age was 25.0 years, and their height,

weight, blood pressure were 165.8cm, 59.0kg, 116.3/67.8mmHg. Before the treatment, we measured

the baseline data (pretest: TB), and after hearing white noise as a loading the stressor, we measured

the stress loading data (TLS). After loading the stressor, we measured the initial data(T0) before

experimental treatment and then we gave ginseng extracts inhalation to the experimental group,

measured the posttest data (T5, T10, T15, T20, T25, T30) every 5 minutes during 30 minutes. Data were

analyzed by t-test, repeated measures ANOVA. Results General characteristics of subjects showed no

difference between two groups. After experimental treatment, the perceived stress, blood pressure,

SPO2 were not significantly different between experimental group and control group. But the BIS

index of experimental group was lower than the control group (F = 14.02, p = .001). Conclusions

Ginseng extract was effective to lower the BIS index although not mitigate the perceived stress level.

Keywords: Ginseng, Stress, Bispectral Index Monitor, Blood pressure

Introduction

Stress is the physiological, physical and psychological reaction to pressure to adapt to difficult

situations. Chronic stress may lead to physical illness such as heart disease, stomach ulcers, high blood

pressure, as well as psychological dysfunctions such as insomnia, neuroses, and depression. Acute stress

reactions include physiological changes, such as elevated blood pressure and increased heart rate caused

by catecholamine production and activation of the sympathetic nervous system, as well as psychological

changes including anxiety. From a medical perspective, stress may cause various diseases; paradoxically,

it is also caused by hospital stays and surgery due to disease. Therefore, reducing patient stress is the

most fundamental responsibility of medical staff. Medical and nursing interventions as well as

complementary and alternative medicine to reduce stress have been continuously developed. Efforts to

reduce stress have also led to the development of integrative medicine approaches that combine

complementary and alternative methodologies with western medicine. Conventional approaches to mitigate

stress rely on drugs such as hypnotic sedatives (Kamata et al., 2010), whereas complementary and


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alternative medicine utilize meditation, music therapy, art therapy, aromatherapy, taichi, yoga, and laughter

therapy. Generally, drug therapy is most commonly used in the clinical setting to mitigate stress whereas

complementary and alternative medicine are not widely utilized. Recently, however, interest in, and use of,

complementary and alternative medicine has gradually increased both in the medical field and in the

general public. Approximately 19–63% of people have used complementary and alternative medicine

within and outside of Korea (Choi et al., 1998; Carlson and Krahn, 2006), mostly for pain mitigation,

function and energy enhancement (Carlson and Krahn, 2006), prolonging life, symptom and side-effect

mitigation, strengthening of the immune system, and quality of life improvement (Correa-Velez et al.,

2005). However, only a handful of studies have been conducted on the anti-stress effects of aromatherapy

essential oils (Field et al., 2008, Nord and Belew, 2009, Saiyudthong and Marsden, 2011). Ginseng has

been shown in several studies to have benefits on immune control (Yi-Seong, 2012), anxiety mitigation

(Cha et al., 2004), as well as control of blood glucose and hyperlipidemia in type 2 diabetes. In addition,

ginseng was shown to have an effect of stress reduction in an animal study on chronic immobilization

stress (Kim et al., 2010). Thus, while the stress mitigation effect of ginseng is believed to be significant,

some researchers have argued that stress mitigation effect is mainly due to the method of ginseng intake,

and scientific evidence is still lacking (Lee et al., 2008). Accordingly, the present study aimed to

investigate the effect of ginseng extracts in inpatients in order to rigorously and scientifically assess not

only conventional treatment approaches but also complementary and alternative medicine to mitigate stress.

Ginseng extracts were produced by steam distillation, a common extraction method used for

aromatherapy essential oils. We believed ginseng extracts would show the effects of saponins, as

ginsenoside Rg1 was identified in chemical composition analysis. Inhalation was used for the application

of the ginseng essential oil because this method is easy and fast. Finally, we investigated the anti-stress

effect of ginseng extracts by evaluating subjective measures such as perceived stress as well as objective

measures, including blood pressure (BP), pulse oxygen saturation (SpO2), and bispectral index (BIS

index).

Method and Materials

1. Study design

The present study investigated the effects of inhaled ginseng extracts on BP, SpO2, BIS index, and

subjective assessment of stress in healthy adults. The study was based on a nonequivalent control group

pretest-posttest design, as follows.

Experimental group

↓

↓

↓

↓

Pre-test (TB)

Stressor(TLS)

Pretest(T0) Treatment

Post-test

(T5,T10,T15,T20,T25,T30)

· General

characteristics

· BP

· SpO2

· BIS Index

· Stress(VAS)

· BP

· SpO2

· BIS Index

· Stress(VAS)

Ginseng

extracts

Inhalation

· BP

· SpO2

· BIS Index

· Stress(VAS)

↑

↑

↑

Control group

<Fig 1. Study design>


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2. Study participants

1) Selection criteria

Study participants were recruited among adults living in D City. All participants signed a written consent form after

receiving full explanations of the study objectives and procedures. The selection criteria used to screen participants are

those who understood the study objectives and voluntarily agreed to participate in the study, and who were not taking

drugs that could affect BP, SpO2, or BIS index. The exclusion criteria are those with chronic disease or diseases that

could affect olfactory function, including sinus infection, asthma, and cold.

2) Sample size calculation

The sample size was calculated by using G power 3.1.7, with the assumptions of a level of 0.05, power of 0.8, an

effect size of 0.25, two groups, and three or more repeated measures in repeated measures analysis of variance

(ANOVA). Under these assumptions, the minimal sample size was a total of 28 participants, with 14 per group. We

recruited additional participants to account for participant drop-out. The final sample included 20 and 25 participants in

the control and ginseng essential oil inhalation (experimental) groups, respectively.

3) Group assignment

Participants were recruited through a recruitment announcement and randomly allocated either to the control or

experimental groups. To minimize treatment diffusion effects during the data collection period, data were collected in

the order of the control group followed by the experimental group. After group assignment, participants were not

provided with any information about their assigned groups.

3. Research instruments

1) Perceived stress : A visual analogue scale (VAS) was used to measure perceived stress. Participants were asked to

select a point on a 0-to-10 scale drawn on a line with 11 numbers to indicate their perceived stress level. Higher scores

indicated higher levels of perceived stress.

2) SpO2: SpO2was measured using an automatic pulse oximeter (Philips M8004A, Germany) placed on the right

index finger. The normal SpO2 range is 97–100%, with levels below 90% indicating hypoxia.

3) BP : BP was measured on the left brachial artery using a module available in a patient monitor (Philips M8004A,

Germany). Participants were placed in a supine position while systolic and diastolic BP were automatically measured

in mmHg.

4) BIS index : The BIS index was measured using a BIS monitor (Aspect Medical System, USA) and sensor

(QUATRO sensor, Aspect Medical System Inc., USA). BIS index values range from 0 to 100; values between 0 and

39 indicate a deep anesthetic state, values between 40 and 59 indicate an intermediate state, values between 60 and 69

indicate a slightly anesthetic state, values between 70 and 79 indicate a pseudo-anesthetic state, value between 80 and

89 indicate a slightly sedated state, and values between 90 and 99 indicate an awakened state.

4. Methods and Procedures

1) Pre-test (TB)

Upon arriving at the lab, participants completed a survey designed to collect their general characteristics

as well as a VAS to indicate their perceived levels of stress. For physiological measurements, the

participants were asked to rest in a supine position for five minutes, at the end of which the measurement


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apparatus were attached to measure BP, SpO2, and BIS index.

2) White noise stimulation (TLS) as a loading the stressor

White noise was administered for five minutes through a headphone set (Philips SHL-1700). Four

minutes after white noise loading, BP, SpO2, BIS index, and perceived stress were measured.

3) Treatment

The experimental treatment was inhalation of ginseng extracts. The ginseng extracts were ginseng

essential oil and ginseng water produced using a patented method (Patent application serial no. 10-2010-

0021948; patent title: Ginseng Extracts - Methods for manufacturing Ginseng essential oil, Ginseng

water and Ginseng essential oil, Ginseng water manufactured by the method). In the ginseng extraction

method, ginseng flowers, seeds, and leaves are washed and steamed, or simply steam-distilled, to

volatilize saponins with steam, which are liquefied in a freezer, which will cause ginseng essential oil and

ginseng water to float. The patent identified various components of ginseng extracts, including

ginsenoside Rg1, a member of a class of saponins. Thus, ginseng essential oil and ginseng water contain

saponins, which are believed to be the components responsible for various benefits.

Following the white noise stimulation, the experimental group rested before they were administered

the experimental treatment in which they were asked to inhale ginseng extracts for 30 minutes. For the

treatment, 0.2 cc of ginseng extracts extracted by steam distillation and stored in a refrigerator were

injected into a partial rebreathing mask. The control group rested for 30 minutes in the supine position

after white noise stimulation. The lab environment was maintained at an interior temperature of 22–25°C

and a humidity level of 40–60%.

4) Post-test (T5,T10,T15,T20,T25,T30)

After the experimental treatment, BP, SpO2, and BIS index were measured at five-minute intervals for

30 minutes. Finally, participants indicated their perceived stress levels using the VAS.

5. Data collection

Data were collected between November 2013 and April 2014. Participants were recruited through an

announcement and screened using the aforementioned selection criteria. All participants received an

explanation of the study objectives and procedures, voluntary participation, and the right to withdraw

from the study at any time, and provided their written consent. Groups were assigned using a random

number generator in Excel to randomly allocate participants into either the control or experimental

groups. To prevent treatment contamination and treatment diffusion, data were collected first for the

control group and then for the experimental group.

6. Data analysis

Between-group homogeneity were assessed using Χ² and t-tests. Additionally, t-tests and repeated

measures ANOVA were performed, and Greenhouse-Geisser correction used if the sphericity assumption of

repeated measures ANOVA was not met.

Results

The general characteristics of the participants did not differ significantly between the experimental and control

groups. After the experimental treatment, perceived stress, BP, and SPO2 did not differ significantly between groups

<Table 1>.


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<Table 1> Homogeneity of General & Characteristics between Experimental and Control group. (N=45)

Characteristics Category Exp. G. (n=25) Cont. G. (n=20)

Χ² p N(%) N(%)

Gender male 9(36.0) 7(35.0) 0.94 1.00

female 16(64.0) 13(65.0)

Mean±SD Mean±SD t p

Age(yr)

25.9.±3.68 24.0±3.2 1.85 .07

Height(cm)

166.2±7.48 165.3±9.70 0.36 .72

Body weight(kg)

58.7±13.71 59.4±10.99 -0.18 .86

Exp.=Experimental group Cont.=Control group SBP=Systolic blood pressure DBP=Diastolic blood pressure

Mauchly's sphericity test showed that the sphericity assumption was not met, based on the repeated

measures ANOVA on perceived stress; therefore, the Greenhouse-Geisser procedure was used. Neither the

interaction between time and group nor between-group differences were significant, but time had a significant effect

on perceived stress (Greenhouse-Geisser F = 60.76, p < 0.001) <Table 2>.

<Table 2> Stress(VAS) between Experimental and Control group. (N=45)

Stress(VAS) Exp. (n=25) Cont. (n=20)

t p F (p)* Mean±SD Mean±SD

Baseline Stress (TB) 5.6±2.24 6.3±1.45 -1.25 .22 Time 60.76

(<.001)

G*T 0.320

(.678)

Group 0.975

(.329)

Stressor (TLS) 7.3±1.85 7.7±1.28 -0.89 .37

Post Stress at 30min (T30) 4.4±2.53 4.6±1.81 -0.43 .67

Exp.=Experimental group Cont.=Control group Mean±SD : Mean ± Standard Deviation *Repeated Measures ANOVA

G*T: Group*Time TB: Baseline TLS: Loading stressor T30: 30min after Experimental Treatment

The repeated measures ANOVA on systolic and diastolic BP also showed that the sphericity assumption

was not met based on Mauchly's test. Neither the interaction between time and group nor between-group differences

were significant, but time had a significant effect on both systolic and diastolic BP (Greenhouse-Geisser F = 7.08, p <

0.001; Greenhouse–Geisser F = 3.21, p = 0.015) <Fig 2>.


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60

<Fig 2. Comparison of Blood Pressure> <Fig 3. Comparison of SpO2>

Repeated measures ANOVA of SpO2 showed a significant interaction between time and group (Greenhouse-

Geisser F = 3.20, p = 0.008) <Fig 3>. In conclusion, ginseng extracts did not appear to mitigate perceived stress,

but did decrease BIS index. Because the repeated measures ANOVA on BIS index showed the sphericity

assumption was not met based on Mauchly's test, the Greenhouse-Geisser procedure was used. The interaction

between time and group (Greenhouse-Geisser F = 3.90, p = 0.009), and the effects of time (Greenhouse-Geisser F = 38.60, p < 0.001) and group (F = 14.02, p = 0.001) were significant <Table 3>.

<Table 3> BIS Index between Experimental and Control group. (N=45)

BIS Index Exp. (n=25) Cont. (n=20)

t p F (p)* Mean±SD Mean±SD

BaselineBIS Index(TB) 93.5±2.97 93.3±3.13 0.24 .81

Time 38.60

(<.001)

G*T 3.90

(.009)

Group 14.02

(.001)

Stressor(TLS) 94.2±2.96 94.6±4.33 -0.33 .74

Post Stress at 0min (T0) 94.0±3.29 92.7±4.16 1.21 .23


Post Stress at 10min (T10) 84.2±4.28 90.9±4.28 -5.19 <.001

Post Stress at 15min (T15) 81.1±5.73 87.4±4.78 -3.92 <.001




Exp.=Experimental group Cont.=Control group Mean±SD : Mean ± Standard Deviation *Repeated Measures ANOVA

G*T: Group*Time TB: Baseline TLS: Loading stressor T0: 0min after Experimental Treatment

T5: 5min after Experimental Treatment T10: 10min after Experimental Treatment




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Discussion

The present study tested the effects of inhaled ginseng extracts on perceived stress (VAS), SpO2, BP, and sedation

level (BIS index). Before the experimental treatment, stress was induced by exposing all participants to white noise.

Then, the experimental group inhaled ginseng extracts, whereas the control group did not. Perceived stress (VAS) was

measured a total of three times to examine change over time; i.e., at the start of the experiment, following white noise

stimulation, and 30 minutes after the experimental treatment. In addition, SpO2, BP, and sedation level (BIS index)

were measured a total of nine times to examine change over time; i.e., at the start of the experiment; following white

noise stimulation; before the experimental treatment; and 5, 10, 15, 20, 25, and 30 minutes after the experimental

treatment.

To investigate the effect of inhaled ginseng extracts on perceived stress, stress levels were measured using the VAS.

The levels of perceived stress (VAS), BP, and SpO2 decreased 30 minutes after the experimental treatment, compared

to levels measured immediately after exposure to the stressor, but the decreases were not statistically significant.

However, sedation level (BIS index) significantly decreased 30 minutes after the experimental treatment compared

to immediately after exposure to the stressor. The BIS index of experimental group was lower than the control

group.

This finding indicates that ginseng essential extracts had a sedative effect on body and mind after exposure to

stress (Lee B et al., 2011). As there are few studies on ginseng and the present study is an early experimental study

on ginseng extracts, it was not possible to perform a comparative analysis or confirm the study finding. Although

only a handful of studies on ginseng exist, ginseng has been reported to reduce anxiety (Zhao Z et al., 2014), as

well as BP at an early stage (Hur MH et al., 2010). Studies on aroma essential oils (Oh J et al., 2008 ; Seong K.,

2013) have reported that treatment with essential oils significantly decreased perceived stress (VAS) and BP;

however, in the present study, ginseng extracts did not significantly decrease the levels of those measures.

Nonetheless, the potential may exist, given that there was a decreasing trend in perceived stress (VAS) and BP.

Thus, additional studies with a more participants are necessary to identify the effect of inhaled ginseng essential

oil on perceived stress (VAS) and BP. Until recently, research on stress and BP has been actively conducted using

various complementary and alternative medicine approaches (Smith JM. et al., 2011). Considering the study

findings from the viewpoint of nursing intervention, perceived stress (VAS) and BP showed a decreasing, and yet

statistically insignificant, trend from white noise exposure to 30 minutes later, while the decrease in sedation level

(BIS index) during the same period of time was significant. Ginseng extracts were effective in lowering the BIS

index, although they did not mitigate perceived stress.

Acknowledgments This research was supported by Basic Science Research Program through the National Research

Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF-

2012R1A1A3013176)


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GINSENG RESEARCH, pp. 256-263.


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SOYBEAN MEAL EXTRACTS WITH HIGH BINDING CAPACITY TO SALMONELLA

Jenifer Mota Rodrigues 1,2

, Maria Eugênia Rabello Duarte2, Miguel Daniel Noseda

2*

1Postgraduate Program in Bioprocess Engineering and Biotechnology and

2Biochemistry and Molecular

Biology Dept., Federal University of Parana, Curitiba, Paraná, Brazil

ABSTRACT: Salmonellosis is a common foodborne infection, reaching almost 100 million cases per

year around the world. Antibiotics are used to control enteric infections, but their prolonged use leads to

the emergence of resistant strains. Researches for alternatives to antibiotics become urgent for health,

industry and livestock sectors. The present study evaluated the capability of soybean meal extracts to

adhere enterobacteria. For this, a soybean meal suspension was subjected to sequential thermochemical

treatments. All the fractions obtained were submitted to chemical and spectroscopic analysis and were

evaluated in vitro for their ability to adhere to Salmonella. All tested products adhered significantly more

bacteria than the negative control and presented similar activities to the positive control, showing a

binding capability between 3.83 x 104 and 6.21 x 10

4 CFU per 3 mg of sample. Fractions with similar

adhesion capacity are chemically distinct; FS-Ep and FS-A are carbohydrate-rich fractions, FS-A, FS-

Tp, FS-Es are protein-rich fractions. All fractions showed galactose, arabinose and galacturonic acid as

main monosaccharides. The diverse chemical compositions of the fractions indicate that other factors or

biomolecules, in addition to mannose, may also be participating in the adhesion process. Results obtained

in this study indicate the biotechnological potential of the soybean meal extracts in the prevention of

enteric infections, as an alternative to the use of antibiotics in livestock.

Keywords: Soybean Meal; Enteric Infections; Enterobacteria; Bacterial adhesion.

1. INTRODUCTION

Enteric infections, mainly transmitted by contaminated food-production animals, are one of the global

morbidity and mortality causes. It is estimated a total of 94 million cases of nontyphoidal Salmonella

infections per year all around the world, resulting in approximately 155 thousand deaths (WHO 2014).

Antibiotics have been extensively used to control these pathogens in animals (FDA 2014).

Additionally, substantial portions of antibiotics have been used for nontherapeutic purposes as growth

promoters (Laxminarayan et al. 2013). This prolonged and excessive use of antibiotics leads to the

emergence of resistant strains (Gullberg et al. 2011).

Researches on alternatives to the use of antibiotics have been intensified (Cox & Wright 2013).

Several alternatives to antibiotics have been proposed, including the use of carbohydrates and

glycoconjugates from different origins (Ofek et al. 2003). These molecules can act as analogue receptors,

mimicking the host receptors located in the intestinal mucosa, leading to modulation of microbiota by

blocking the pathogen adhesion to the tissue. Thus, strategies using anti-adhesion agents can be effective

in reducing infections, diseases and the risks of transmission (Becker et al. 2007). Furthermore, anti-

adhesion therapy appears to be safer, softer and sustainable with the added benefit of minimizing selective

pressure because the pathogens are prevented from binding to host cells rather than being killed (Thomas

2010).

Yeast derivatives have been sold worldwide, as anti-adhesive products, in the feed supplements market

for a long time (Ganner et al. 2013). However, the true efficacy concerning the use of analog receptors

remains unclear (Gaggia et al. 2010). There are questions about exact mechanisms related to the

anchoring of strains to the enterocytes surface (Bardiau et al. 2010) and more informations are needed to

elucidate the ideal chemical structure involved in bacterial adhesion process.


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Soybean is a versatile grain used by the agribusiness, chemical and food industry. Its main use is oil

extraction, generating large quantities of soybean meal, a by-product of high nutritional value mainly

directed to manufacture animal feed. Anti-adhesive properties of soybean glycopeptides (Neeser et al.

1986), soy flour oligomannosides (Neeser & Wursch 1990) and tempeh (Kiers et al. 2002; Kiers et al.

2003; Becker et al. 2007; Becker & Galletti 2008) have been described. The present study aimed to

evaluate the capability of soybean meal extracts to adhere enterobacteria Salmonella enterica sv.

Typhimurium, using in vitro assays.

2. MATERIALS AND METHODS

2.1 Isolation of Soybean Meal Extracts:

Soybean meal (Import, Export and Oil Industry S.A, Brazil) was extracted with distilled water (150 g L-1

)

at pH 2.0, 120 °C, 1 atm for 1 h (thermochemical process). After filtration with fine cloth, neutralization

and centrifugation the supernatant was subjected to a second thermochemical process (15 min). The

treated material was then concentrated to 30% of its original volume and a third of this concentrated

solution was dialyzed (Spectra/Por® Dialysis Tubing, cut-off 6-8 kDa, Spectrum Laboratories Inc.) and

freeze-dried, yielding FS-A fraction (7.9% yield). The remaining solution was then deproteinized with

13.3% trichloroacetic acid (w/v). After 10 min under magnetic stirring, the suspension was centrifuged

and the precipitate was redissolved in distilled water, neutralized, dialyzed (cut-off 6-8 kDa) and freeze-

dried, yielding FS-Tp fraction (2.8% yield). The supernatant was added to excess absolute ethanol (3:1

v/v) and maintained for 18 h at 4°C. The supernatant and precipitate, obtained by centrifugation, were

neutralized, dialyzed (cut-off 6-8 kDa) and then freeze-dried to give FS-Es (0.9% yield) and FS-Ep

fraction (2.5% yield), respectively.

2.2 Analytical Methods:

Total carbohydrates, uronic acids and proteins contents were determined according to DuBois et al.

(1956), Filisetti-Cozzi & Carpita (1991) and Lowry et al. (1951), respectively, using galactose,

galacturonic acid and bovine serum albumin as standards. For determination of monosaccharide

composition, the fractions were hydrolyzed (2 M trifluoroacetic acid, 100ºC, 6 h), reduced with sodium

borohydride and acetylated with acetic anhydride (120ºC, 1 h). The alditol acetates derivatives were

analyzed by GC-MS using a Varian 3800 chromatograph coupled to a Varian Saturn 2000R ITD

spectrometer with a DB-225 capillary column (30 m x 0.25 mm i.d.) programmed from 50°C to 220°C at

40°C min-1

, using helium as carrier gas (1 mL min−1

). The products were identified by their typical

retention times and electron impact profiles (Jansson et al. 1976).

2.3 Nuclear Magnetic Resonance Spectroscopy (NMR):

NMR spectroscopic analyses were carried out using a Bruker Avance III 400 MHz spectrometer (Bruker

BioSpin Corporation) equipped with a 5 mm inverse probe. 13

C acquisition parameters were previously

reported by Ascêncio et al. (2006). Analyses were carried out at 70°C on samples dissolved in D2O at 80

mg mL−1

. Chemical shifts are expressed in ppm relative to internal acetone standard at 30.2 ppm. NMR

spectra of insoluble fractions were acquired using the 13

C CP/MAS (cross-polarization/magic angle

spinning) solid-state NMR technique in a Bruker Avance 400 MHz spectrometer operating at 9.4 T and

frequency of 100.62 MHz, equipped with a 4 mm MAS probe. Samples were spun at about 5 kHz.

Contact time, acquisition time and recycling time were 2 ms, 0.03 s and 5 s, respectively. The spectral

width was 26 kHz, with 4 k data points and the number of scans 1 k.

2.4 In vitro Adhesion Assay.


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(i) Principle: Bacterial strain was allowed to adhere to the tested samples supplied as well

coatings in microplates in a miniaturized adhesion assay, adapting a method described by Becker et al.

(2007) and Ganner et al. (2010). This method evaluates in vitro adhesion of bacteria on samples by

measuring the optical densities (ODs) of the culture solutions, which indicate the growth of the adhering

bacteria. There is an inverse relationship between initial cell densities (number of adhering bacteria) and

the growth rate: the higher the adhering cell numbers, the shorter the detection times of growth.

(ii) Bacterial Strain and Culture Conditions: Bacterial strain used was Salmonella enterica sv.

Typhimurium ATCC 14028 maintained in Brain Heart Infusion Agar (HiMedia Laboratories Pvt. Ltd)

under aerobic conditions and stored for not more than 2 weeks at 4°C. Bacteria were transferred to

Tryptic Soy Broth (TSB, HiMedia Laboratories Pvt. Ltd) and subcultured in the same medium at 37°C to

log phase growth for 18 h under aerobic conditions. The OD of bacterial suspension was adjusted to 0.01

(at 690 nm) for the experiments as described by Ganner et al. (2010).

(iii) Calibration Curve: Serial dilutions of freshly prepared cultures were plated, in triplicate, on

Tryptic Soy Agar and incubated for 24 h, prior to CFU enumeration. Simultaneously, eight replicates of

each bacterial suspension were incubated, 200 µL per well in a microplate reader (SpectraMax 340;

Molecular Devices Ltd) for 18 h at 37°C for determination of growth parameters. The ODs (at 690 nm)

were determined at intervals of 15 min, with automatic shaken at medium intensity for 3 s prior to every

reading. Calibration curve (linear regression) was produced as described earlier by Ganner et al. (2010),

plotting the time in hours when the adhering bacterial solution reached the exponential phase (OD of 0.1

at 690 nm) versus the number of counted bacteria on the agar plate (CFU mL-1

), in order to calculate

viable numbers of bacterial cells bound to the sample.

(iv) Assay: The samples tested were suspended in phosphate buffered saline (PBS: 10 mmol L−1

sodium phosphate, 0.15 mol L−1

NaCl, pH 7.0) to a final concentration of 1% (w/v) and homogenized in

an ultrasonic bath three times for 30 s each. For coating, 300 µL/well of the suspensions were pipetted in

a high binding polystyrene microtitration plate (Costar 3590, Corning) and incubated for 16-18 h at 4°C.

The plate was washed three times with 300 µL/well with PBS to remove unbound material. Subsequently,

the wells were blocked with 300 μL of 1% BSA (Bovine Serum Albumin, Sigma Aldrich) in PBS (w⁄v)

for 1 h at 4°C. Then, the plate was again washed three times with PBS. Thereafter, 300 µL/well of

bacterial suspension OD 0.01 was added to the microtitration plate. Bacteria were allowed to adhere to

samples for 1 h at 37°C. Afterwards, the wells were washed 6 times with 300 µL/well of PBS to remove

non-adherent bacteria. The wells were filled with 200 μL of TSB and one drop of paraffin-oil was added.

The microtitration plate was placed in a microplate reader and bacterial growth was monitored by OD

(690 nm) at 37°C, for 16 h at intervals of 15 min, with automatic shaken at medium intensity for 3 s prior

to every reading. All readings were performed in three independent assays and in triplicate per assay.

Growth control, binding control, positive control, media control and blank samples were also assessed

in each plate. The growth control consisted of non-coated well with BSA containing a suspension of

bacteria in TSB with OD 0.01. The binding control consisted of BSA and bacteria but without adding

sample to determine the non-specific binding. A commercially available yeast cell wall-derived product,

commonly applied in the feedstock industry to control zoonoses, was used as positive control. The blank

consisted of the sample and BSA but without adding bacteria. The blank and media control showed no

growth until after 16 h and negative control had no growth until after 5 h.

(v) Data Handling and Statistics: The data generated by the photometer software (Soft-MaxPro

version 2.2.1.; Molecular Devices Ltd) were converted into text files and analyzed using R Software


(978-0-9951985-1-7)

66

(version 2.15.1; The R Foundation for Statistical Computing). All OD data were processed by nonlinear

regression analysis employing a bacterial kinetic model described by Becker et al. (2007):

⁄ ⁄ (1)

where, fOD (t) denotes the optical density of the liquid medium at time t after incubation. A represents the

asymptotic optical density in the stationary phase, B is the time after incubation at which half of the

asymptotic optical density has been reached, and C is a constant determining the sharpness of the

switching characteristic of the curve. Using this sigmoidal equation, the tOD parameter was determined,

representing the time at which an OD690 of 0.1 was reached at the threshold of visible growth:

[ ]

⁄ (2)

All tOD values were transformed into log CFU ml-1

by means of linear regression fitted to the

correlations between OD and colony counts (see calibration curve), thereby determining the quantity of

bacteria originally connected to the extracts studied (Ganner et al. 2010). Additionally, samples were

compared to the positive control, establishing the number of bacteria attached to the control as 100%. The

tOD parameter was statistically evaluated applying analysis of variance (ANOVA). Differences between

mean values were tested using Tukey’s test. Data with a p value less than 0.05 was considered statistically

significant.

3. RESULTS AND DISCUSSION

Several studies have shown the promising effects of carbohydrates and glycoconjugates from different

origins with potential to inhibit the adhesion of pathogens, such as Salmonella, Campylobacter or E. coli

to the intestinal mucosa of animal species. The similar nature of these molecules with the intestinal

receptors of such pathogens provides an alternative adhesion matrix to enteropathogenic bacteria. Table 1

gives an overview of the results, presenting detection times of growth for S. enterica sv. Typhimurium in

microplates coated with soybean meal extracts. The correlation between time and initial cell density

shows the adhesion ability of the tested molecules. The higher the adhesion potential, the greater the

number of bacteria initially bonded to the molecules, the faster the bacterial growth and the shorter the

time required reaching OD of 0.1. All tested products adhered bacteria significantly more than the

negative control (Table 1). The difference in tOD values between the tested samples and the negative

control were 1.92 h for FS-Es, 1.75 h for FS-Tp, 1.74 h for FS-Ep, 1.69 h for FS-A and 1.22 h for the

positive control. FS-Es fraction presents significantly higher adhesion capacity than the positive control.

The other fractions presented statistically similar potential to the positive control. All fractions were

analyzed on three independent assays in triplicate, using the same inoculum batch to minimize the

coefficient of variation (CV) of the tests. The CV average intra and inter assays for the tOD was

approximately 8.8%, value close to that found by Ganner et al. (2010).

From the growth parameters obtained for the serial dilutions of bacterial suspension a linear regression

was compiled (y = -0.911x + 8.322) to calculate the unknown amount of bound bacteria to samples

depending on the detection time. The R² for fitting of the regression was 0.996, indicating an excellent fit.

Thus, the fraction with higher adhesion capacity, FS-Es, was able to adhere 6.21 x 104 CFU per 3 mg of

sample (Table 1). The remaining investigated fractions, showed a binding capability between 3.83 x 104

and 4.41 x 104 CFU/3 mg of sample. Comparing the mean number of adhered bacteria of each fraction

with the commercial product, it is verified that FS-Es fraction adhered 431.3% more bacteria than the

positive control, while the other fractions adhered between 266 and 306.3%.

The soybean meal fractions were analyzed by chemical (Table 1) and spectroscopic methods (Figure

1). Although these fractions have similar adhesion capacity, they are chemically distinct. FS-Es (ethanolic

supernatant) and FS-Tp (TCA precipitate) have a high protein content (58.0 and 86.8%, respectively), and

low carbohydrate content (8.7 and 21.2%, respectively). Conversely, FS-Ep fraction (ethanolic


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67

precipitate) has a high carbohydrate content (82.7%) and low protein (4.7%). The aqueous extract FS-A,

in contrast, has comparable amounts of protein and carbohydrates, 40.1 and 42.6%, respectively. The

obtained fractions, in common have arabinose, galactose and galacturonic acid as major monosaccharide

constituents. Glucose and mannose appear in significant quantities in FS-Tp fraction (Table 1).

Table 1 – Detection times of bacterial growth tOD (h) for Salmonella enterica as a measure of adhesion to

soybean meal extracts, chemical analyses and monosaccharide composition of the tested fractions

Samplesa tOD=0.1 (h)

b CFU/ 3

mgc

% C+

relationship PTN (%)

d CHO (%)

e

Monosaccharides (mol%)f

Rha Fuc Ara Xyl Man Gal Glc GalA

FS-Es 3.87C 6.21 x 10

4 431.3 58.0 21.2 1.3 tr 10.2 tr tr 2.6 tr 85.8

FS-Tp 4.04B-C

4.41 x 104 306.3 86.8 8.7 1.8 2.8 13.9 2.9 22.9 5.6 21.3 28.7

FS-Ep 4.05B-C

4.33 x 104 300.7 4.7 82.7 8.1 tr 15.1 5.8 2.1 28.2 6.8 33.9

FS-A 4.10B-C

3.83 x 104 266.0 40.1 42.6 tr tr 26.4 3.7 tr 50.4 4.3 15.2

C+ 4.57B 1.44 x 10

4 100.0 - - - - - - - - - -

C- 5.79A 1.11 x 10

3 7.7 - - - - - - - - - -

a Samples defined in the text;

b Mean values within a column differ significantly (p<0.05).

c Amount of

bound bacteria determined by linear regression; d PTN: Percentage of proteins;

e CHO: Percentage of total

carbohydrates; f Monosaccharides quantified in mol% in the acetate alditol form. C+: positive control; C-:

negative control; Rha: rhamnose; Fuc: fucose; Ara: arabinose; Xyl: xylose; Man: mannose; Gal:

galactose; Glc: glucose; GalA: galacturonic acid; tr: percentages lower than 1%.

The 13

C NMR spectra of the studied fractions are shown in Figure 1. Characteristic protein signals

were identified in the NMR spectra of FS-A and FS-Es fractions (Figure 1a and 1c) and 13

C CP/MAS

NMR spectrum of FS-Tp fraction (Figure 1d). The signals between 185-165 ppm correspond to the

carbonyl carbon of the main chain and carboxylic groups of the side chain of acidic amino acids; signals

between 135-115 ppm were assigned to the side chain carbons of aromatic amino acids; resonances

between 65-45 ppm and 45-25 ppm were assigned to and , carbons, respectively; and the resonances

between 25-15 ppm correspond to the methylene and methyl groups of the aliphatic side chains (Ma et al.

2015).


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68

Figure 1 - 13

C NMR spectra of FS-A (a), FS-Ep (b), FS-Es (c) and 13

C CP/MAS NMR spectrum of FS-Tp

(d). Insets: anomeric region of the corresponding fractions.

In addition to the protein signals, FS-A, FS-Es and FS-Ep spectra show intense resonances between

110 and 60 ppm, characteristic region of carbohydrates. The anomeric region of FS-A and FS-Ep spectra

(Figure 1a' and 1b') shows an intense signal at 104.4 ppm corresponding to C-1 of β-D-Galp units

(Fransen et al. 2000). In this fraction, are also observed signals at 77.6, 74.5, 73.5, 72.1 and 60.9 ppm,

corresponding to C-4, C-5, C-3, C-2 and C-6, respectively, of the β-D-Galp-(14)-linked units (Fransen

et al. 2000; Cipriani et al. 2009).

In the 13

C NMR spectra of FS-A and FS-Es fractions we can additionally observe resonances at 107.7,

107.3 and 106.5 ppm assigned to the C-1 of α-L-Araf units (Fransen et al. 2000). Resonances at 100.1 and

99.3 ppm, correspond to C-1 of esterified and non-esterified α-D-GalpA units (Cozzolino et al. 2006), and

at 173.4 and 52.8 ppm to free carboxylic and methyl ester groups of GalA units, respectively (Delgobo et

al. 1999; Cozzolino et al. 2006).

Correlation of the adhesion capacity results with chemical composition of the tested fractions, suggests

that both, proteins and carbohydrates, are involved in the adhesion mechanism, since protein-rich

fractions (FS-Tp and FS-Es), and carbohydrate-rich fractions (FS-Ep) presented a high adherence

capacity.

The pathogenesis of Salmonella enterica infections requires adherence to various surfaces of the host

cells and a large number of adhesive structures may be involved, however, the full functionality of these

structures is still not completely elucidated (Yue et al. 2012; Fàbrega & Vila 2013). Among the different

types of fimbriae described for serovar Typhimurium, type I is reported as major contributor for the

fixation on enterocytes and colonization of the intestine (Kisiela et al. 2011). FimH, a subunit of type I

fimbriae, represents the fimbrial lectin indispensable for the binding to intestinal epithelium. This lectin is

highly specific for mannose residues, however, studies have shown that allelic variants of FimH,

commonly found in serovar Typhimurium, can be responsible for changes in the binding properties of

these fimbriae, leading to affinity changes or even specific changes (Yue et al. 2012). Moreover, the

adhesion process may involve other factors beyond the adhesin-receptor interactions, such as hydrophobic

interactions or non-specific binding in different levels of strength (Ofek et al. 2003).

Neeser et al. (1986), Neeser & Wursch (1990), Kiers et al. (2002), Kiers et al. (2003), Becker et al.

(2007) and Becker & Galletti (2008) have shown the ability of soy and derivatives to bind Enterobacteria,

as Salmonella sp. and E. coli, avoiding the subsequent tissue colonization by the pathogen. The

demonstrated binding ability for soy was correlated with the presence of mannose in the extracts and their

binding to bacterial lectins. Among the studied fractions, only FS-Tp contains mannose in a significant

content, 22.9 mol%. However its absolute content is low, corresponding to 2 mol%. It is possible that

mannose, even at low levels, may be acting as analog receptor due to the high specificity of this sugar. In

addition, bacteria do not only recognize carbohydrates in reducing terminals, but also in internal links

(Sharon & Ofek 2000), so mannose residues may be recognized in chains mainly composed by other

monosaccharides.

Bouckaert et al. (2005) analyzed the affinity of E. coli FimH adhesins against different

monosaccharides and disaccharides. Their results showed that FimH binds preferentially to mannose but

can also bind to other saccharides, such as fructose and glucose, with lower affinity. Previous studies

carried out by Old (1972), Salit & Gotschlich (1977), Korhonen et al. (1980), Ashkenazi & Mirelman

(1984), Sanchez et al. (1993) and Spring et al. (2000) have already demonstrated adherence capacity of

different Salmonella species and E. coli to these same monosaccharides. Most fractions tested in the

present study, show glucose in their composition, with absolute levels ranging between 1.8 and 5.6%,

suggesting that glucose may be involved in the adhesion process of bacteria to soybean extracts.


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69

Besides mannose and glucose, other monosaccharides may be interacting with Salmonella adhesins.

All the bioactive fractions obtained from soybean meal presented in its composition galactose, arabinose

and galacturonic acid. There are reports of inhibition of Salmonella adhesion by D-Galactose and L-

Arabinose (McHan et al. 1989; Oyofo et al. 1989; Chessa et al. 2009; Parker et al. 2010; Sakarya et al.

2010), galactooligosaccharides (Tzortzis et al. 2005; Searle et al. 2009; Searle et al. 2010) and pectic

polysaccharides obtained from kiwi and plantain (Parkar et al. 2010; Roberts et al. 2013).

In addition to carbohydrates, there are reports of reduction of adhesion by -conglycinin, a

glycopeptide highly present in soy (Yang et al. 2008). Shoaf-Sweeney & Hutkins (2008) proposed that

the adhesion affinity is defined by the valence of the protein-carbohydrate interactions; an isolated

carbohydrate molecule has lower affinity than a complex structure involving protein and different glycan

units. The fractions that have higher adhesion capability, have high protein contents (FS-Es: 58%; FS-Tp:

86.8%), indicating that there may be some interaction between the proteins and adhesins. Proteins can

also be acting synergistically with carbohydrates.

The carbohydrates are related to the future of anti-adherent therapy. The present study highlights the

potential of different soybean meal extracts as alternative binding matrices for enteropathogens and their

biotechnological potential in the prevention of enteric infections, and as an alternative to the use of

antibiotics in livestock. Additionally, the diverse chemical composition of the fractions clearly indicates

that the presence of mannose cannot be the only factor responsible for the binding of bacteria to the

soybean meal extracts, other factors or biomolecules may also be participating in the adhesion process.

However, the exact mechanism of the binding remains to be further elucidated.

ACKNOWLEDGMENTS

This study was supported by grants from CNPq (Universal Calls: 485980/2012-6 and 462414/2014-0).

The authors are grateful to Professor Wagner Bonat for the help with calculations from sigmoidal model

and to Bio4-Soluções Biotecnológicas for supplying soybean meals. M.D.N. and M.E.D. are Research

Members of the National Research Council of Brazil (CNPq). J.M.R. acknowledges a doctoral

scholarship from CAPES and postdoctoral fellowship from CNPq.

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