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www.nutri-facts.org

Agro FOOD Industry Hi Techvol 25(5), September/October 2014

COVER STORY

2 The forgotten nutrient? Vitamin E and the latest science

EDITORIAL

4 Functional foods and their clinical implications U. N. Das

PRE-PROBIOTICS

6 Healthy gut microbiota and long term health Y. Vandenplas

10 Probiotics and Prebiotics to save human microbiota enhancing health and well-being. Note I P. Morganti, U. Cornelli, G. Gazzaniga

INFANT NUTRITION

15 Enrichment of infant formula with docosahexaenoic acid (DHA) and arachidonic acid (ARA)

S. T. Gautier

HEALTHY INGREDIENTS

19 Sensory and instrumental properties of cookies enriched with "vitalplant" - extract M. V. Pestori, A. . Mian, O. D. imurina, D. J. Jambrec, M. M. Belovi, J. M. Gubi, N. M. Nedeljkovi

ANTIOXIDANTS

24 Effects of solvent extraction system on concentration and antioxidant activity of strawberry phenolics M. N. Miti, D. A. Kosti, A. N. Pavlovi, D. S. Dimitrijevic, J. N. Veljkovic

FUNCTIONAL DRINKS

30 Antibacterial activity of donkey milk against SalmonellaL. ari, B. ari, A. Mandi, J. Tomi, A. Torbica, N. Nedeljkovi, B. Ikoni

35 The neurobiology and marketing of mood drinksR. Cheatham

FOOD ANALYSIS

39 Hyperspectral imaging in plant based food safety - Possibility of hyperspectral imaging applicationin safety control of plant based food that is converted to by-products and used as feed

D. olovi, B. Koki, I. abarkapa, J. Levi, O. Djuragi, K. Teichmann, D. Jdrejek

44 Rapid authentication of white wines. - Part 1: Classification by designation of origin M. J. Martelo-Vidal, M. Vzquez

FOOD SAFETY

49 Feed undesirable substances as food contaminants - Part 2: Dioxins P. I. Natskoulis, P. E. Zoiopoulos

SPORT NUTRITION

53 New product development: carbonated beverage with different protein and creatine forsportsmen and physically active people

M. Tomczyska-Mleko

FOOD RISK

57 Traditional and social media in food risk communication M. Friel, J. M. Wills

PRESERVATIVES

61 Effect of modified atmosphere packaging on persimmon fruit (cv. Karaj) - Physical, chemical andmechanical properties

A. Dadashpour, M. Rahemi, M. Jouki

65 NEWS

Agro FOOD Industry Hi Techis a peer-reviewed journaldevoted to functional food and nutraceuticalsaddressed to a readership belonging to the industry.The 2013 impact factor is 0.294, according to theThomson Reuters Journal Citation Reports.

TEKNO SCIENZE SRL

Viale Brianza, 22, 20127 Milano (Italy)

Tel +39-02-26809375/28381260

Fax +39-02-2847226

e-mail: [email protected]

web: www.teknoscienze.com

Editor in ChiefCarla Scesa - [email protected] Director

Silvana Maini - [email protected] EditorsGayle De Maria - [email protected] Weighardt - [email protected] & SalesGiulio Fezzardini - [email protected] Baldina - [email protected] Rivarollo - [email protected] & EventsPaola Passadore - [email protected] ManagerElisa Novaresi - [email protected]

Production AssistantL. A. Alburqueque Diaz - [email protected] & AccountAlba Aprea - [email protected]

PRINTING OFFICE:AGC - Arti Grafiche Colombo(Gessate MI, Italia). Authorization fromTribunale di Milano n. 343 of May 1990.Autorizzazione del Tribunale di Milanon. 343 del Maggio 1990.

SCIENTIFIC ADVISORY BOARD:Anton Alldrick (CCFRA)Sybille Buchwald-Werner (Vital Solutions GmbH)

Undurti N Das (UND Life Sciences)Peter Engel (DSM)Inga Koehler (Analyze & Realize GmbH)Ray A. Matulka (Burdock Group)Vicente Micol (University Miguel Hernndez)Arthur Ouwehand (University of Turku)Demosthenes B. Panagiotakos (Harokopio University)Denis Poncelet (ONIRIS and Capsulae sarl)Igor Pravst (Nutrition Institute Slovenia)Joe Romano (Waters)Yang Zhu (Wageningen University)Ralf Zink (DMK - Deutsches Milchkontor)


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Agro FOOD Industry Hi Techcustomer's publicationCOVER STORY

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

The forgotten nutrient?Vitamin E and the latest science

alone. Indeed, a recent study by the University of Viennafound that vitamin E has low stability in vegetable oils andlevels decline throughout the shelf life of a product as aresult (1). This could be a major factor behind thewidespread vitamin E insufciency that is currently observed

in population groups across the globe.The vitamin E content in food and beverages is oftenreported as a-tocopherol equivalents (a-TE). This term wasestablished to account for the differences in biologicalactivity of the various forms of vitamin E. TheRecommended Daily Allowance (RDA) for adults is 15mgRRR-a-tocopherol/day (FNB, 2000) in the US. In Europe,adult recommendations range from 4 to 25mga-tocopherol equivalents (a-TE)/day for men and from 3 to12mg a-TE/day for women. In fact, there is research tosuggest that the RDA should be raised to 30mg a-TE/day.

The role of vitamin E

The main biological function of vitamin E is as anantioxidant, preventing the propagation of free-radicalreactions and playing a critical role in cell signalling, geneexpression and the regulation of other cell functions. TheEuropean Commission has authorized an Article 13.1 healthclaim stating that vitamin E contributes to the protection ofcell constituents from oxidative damage. The damagingeffects caused by free radical reactions can lead tovarious health conditions such as heart disease, cancerand inammatory conditions.

Until recently, little new scientic effort has been devotedto studying the role of vitamin E in the human body. There ishowever emerging evidence to demonstrate that vitamin E

supplementation can reduce the negative health

JACOB BAULY

Global Marketing ManagerDSM

Vitamin E plays a vital role in supporting brain, eye,cardiovascular, maternal and infant health, as well asprotecting the skin. The importance of vitamin E in humanswas not recognized until relatively late in the history ofvitamins and it is estimated that more than 90 per cent ofthe population in the United States does not meet thedietary intake recommendations for vitamin E.

The outcomes of studies to investigate the possible effect ofvitamin E on cardiovascular disease have been inconsistentand subsequent media coverage has taken attentionaway from the fundamental role that vitamin E plays in

supporting human health. The impact of low vitamin Eintake globally should be a serious public health concernand, while the micronutrient is not currently high on theagenda of scientists and funding organis ations, there is anurgent requirement for additional research.

E is for Essential

Vitamin E is a generic term for eight fat-soluble compoundsfound in nature, of which alpha-tocopherol has thehighest biological activity and is the most abundant in thehuman body. The most important sources of vitamin E arevegetable oils, nuts, wholegrains and wheatgerm and thereis also a limited supply in seeds and green leafy vegetables.Due to changes in modern eating habits, it can be difcult

to obtain the required amount of vitamin E through diet


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3Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

implications of fatty liver disease (2). This is a potentiallysignicant development as obesity-related conditions such

as fatty liver disease and diabetes become key publichealth concerns on a global scale. There is also research tosuggest that vitamin E intake reduces and normalizes therisk of cardiovascular events in a group of diabetic patients,in particular those which have a specic genotype (3). Inaddition, the role of vitamin E supplementation in thetreatment of neurodegenerative diseases such asAlzheimers disease is under investigation (4) and there isongoing research into the role that vitamin E plays inprotecting omega-3 polyunsaturated fatty acids fromoxidative damage (5).

Need for action

Vitamin E is essential as it is a key element in cellmembranes to protect against the damaging effectscaused by oxidation and it offers numerous health benets.DSM has an ongoing commitment to advance researchinto vitamin E and is calling on consumers, healthcareprofessionals, scientists and governments to take note ofemerging science. It is also leading an initiative - Vitamins in

Motion - to highlight the critical role vitamins play in overallnutrition and health. For more information and to accessthe latest news on vitamin E, visit www.dsm.com/vitamin-e.

References

1. Pignitter et al, Cold uorescent light as major inducerof lipid oxidation in soybean oil stored at householdconditions for eight weeks, J Agric Food Chem. Mar12, 62(10), 2297-305 (2014)

2. N. Chalasani et al, The Diagnosis and Management ofNon-Alcoholic Fatty Liver Disease, Heptatology, June2012.

3. S. Yusuf et al, Effects of an Angiotensin-ConvertingEnzyme Inhibitor, Ramipril, on Cardiovascular Events inHigh-Risk Patients, New England Journal of Medicine,Mar 9, 342(10), 748 (2000).

4. M. Dyksen et al, Effect of Vitamin E and Memantine onFunctional Decline in Alzheimer Disease, Journal of the

American Medical Association, 311(1), (2014).5. Food and Nutrition Board, Institute of Medicine Dietaryreference intakes for vitamin C, vitamin E, selenium,and carotenoids, 186 283 (2000), National AcademyPress, Washington.

For more information

DSM Nutritional ProductsExternal Communications

Alex Filztel. +41 61 815 83 67

email: [email protected]


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It is generally agreed that diet may modify the risk of developing or exacerbating certain chronic diseases. This observationis not new since for centuries it is known that diet and foods have a functional role in health. What is new is the scientificevidence as well as the terminology.

Our understanding of the relationships between food, physiological function and disease has progressed in recent yearsespecially, with regard to the role of nutrition in our state of well being. This led many manufacturers to develop and marketproducts derived from foods that could be used to promote good health. This resulted to the development and marketingof a growing spectrum of products called nutraceuticals and functional foods.

Although the terms nutraceutical and functional food are used commonly around the world, there is no consensus ontheir meaning. Bureau of Nutritional Sciences, of the Food Directorate of Health Canada, has proposed the followingdefinitions:

A nutraceutical is a product isolated or purified from foods that is generally sold in medicinal forms not usually associatedwith food. A nutraceutical is demonstrated to have a physiological benefit or provide protection against chronic disease.

A functional foodis similar in appearance to, or may be, a conventional food, is consumed as part of a usual diet, and isdemonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions.

As commercial interest in the marketing of these foods and components grows, regulatory agencies face new challenges.Current understanding of the benefits and risks related to health claims on such products is more qualitative thanquantitative. Hence, regulators need to exhibit caution and explore this issue from a variety of perspectives. For instance, How should nutraceuticals/functional foods be defined with precision for regulatory purposes? Should such products remain as either foods or drugs under the Food and Drugs Act?

What kinds of health claims, if any, should be allowed on food labels? What standards of evidence would be necessary and sufficient to prove a health benefit? How can nutraceuticals/functional foods be regulated without unduly compromising the right of citizens to takegreater responsibility for their own health?

Current regulatory environment is said to discourage innovation and marketing of nutraceuticals/functional foods. Underthe Food and Drugs Act, only a specified range of claims may be made for foods; otherwise, they are classed as drugs. Thefood regulations currently permit: positioning the food as part of healthy eating, claiming that a nutrient or nutritive substance is generally recognized as an aid or factor in maintaining the functions ofthe body, or necessary for the maintenance of good health and normal growth and development (also known asbiological role claims and nutrient function claims).

Under the current regulatory framework nutraceuticals/functional foods appear to have an awkward fit. Although somemay appear to consumers as ordinary foods, they are known to produce physiological effects.

EDITORIAL

UNDURTI N DAS

UND Life Sciences, 2020 S 360th St, # K-202, Federal Way, WA 98003, USA

Member of AgroFood industry Hi Tech Scientific Advisory Board


Functional foodsand their clinical implications


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One needs to note that foods are consumed forwellness whereas drugs are necessary to fightillness. Moreover, drugs need to meet stringentregulations governing Good ManufacturingPractices, testing procedures and post-marketsurveillance. In view of this dichotomy betweenfunctional foods and drugs caution needs to beexercised.

Since, the terminology and definitions fornutraceuticals/functional foods are not consistentworldwide, and there are no international standardsfor regulation of nutraceuticals/functional foods toserve as baselines, it is important that (i) healthclaims on products are truthful and not misleading,(ii) different claims will require various levels ofevidence and the quality of this evidence must beconsistent, (iii) health claims on foods would requirea common scientific basis to provide consistent andcredible messages to the public, (iv) health claimson products should be established in the context ofthe total diet and other relevant lifestyle factors, and

finally (v) the possibility that addition of high doses ofnutrients or other food components to a diet - eitherthrough fortification or non-food sources such asnutrient supplements and over-the-counterpharmaceutical preparations - may result in adversehealth effects that needs to be kept in mind.

In view of these controversies, more research anddiscussion among all stake holders and a commontheme under which labeling claims are agreed uponneeds to be reached. This needs cooperation andcoordination from one and all.

Furthermore, one important question that needs tobe addressed is whether foods that are producedbased on recombinant technology such that theycontain higher amounts of phytochemicals, vitamins,infection resistant chemicals, etc., can be labelledas foods or food medicine or frankly as drugs. This isespecially so in the context of foods that maycontain super amounts of certain nutrients/vitamins/phytochemicals, etc., that may exceed thepermissible amounts of daily recommended amount.Though this is a theoretical possibility, while definingfunctional foods and permitting them to be free lyavailable for human consumption it is important toconsider this possibility. It is also essential that public

is reassured about the safety and usefulness of foodsproduced by recombinant biotechnology. This needsmore scientific studies that are transparent.

An example of functional food components thathave health benefits are given in Table 1. One bigquestion is how long and how much of thesefunctional food components can be consumed byhumans without significant side-effects. Thus, goodpharmacokinetic, phamacodynamic andpharmacovigilance studies and relevanttoxicological studies are needed both for the sakeof science and human health and to ensure thepublic that functional foods and their componentsare safe.


Table 1.Example of functional food components that have health benefits.


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6 Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

KEYWORDS: microbiota, microbiome, gut flora, cesarean section

AbstractThis review summarizes how the composition of the gastro-intestinal microbiota depends on pre- andpostnatal factors, and birth itself. The impact of method of delivery, feeding during infancy andmedications, such as antibiotics and anti-acid medication, on the composition of the gastro-intestinal microbiota has clearly beenshown. However, the duration of the impact of these factors is not well established. The gastro-intestinal microbiome composition isassociated with many auto-immune mediated diseases. Although causality has not been obviously demonstrated, there is a strongtendency in this direction. Nevertheless, results of the manipulation of the gastro-intestinal microbiome composition in these conditionsare often disappointing. A better understanding on factors determining the long-term composition of the gastro-intestinal microbiomeand its health consequences are a priority research topic. A better understanding of the association between the microbiome andthe immune system may have a tremendous impact on general health.

Healthy gut microbiotaand long term health

INTRODUCTION

The microbiome is the ecological community of commensal,symbiotic, and pathogenic microorganisms that literallyshare our body space. This term was originally coinedby Joshua Lederberg, who argued the importance ofmicroorganisms inhabiting the human body in health anddisease. A dysbiosis is condition with microbial imbalanceson or inside the body. Dysbiosis is most prominent inthe digestive tract or on the skin, but can also occur onany exposed surface or mucous membrane such as lungs,mouth, nose, sinuses, ears, nails, or eyes. Dysbiosis has beenassociated with different illnesses, such as gastroenteritis,allergy, inammatory bowel disease, etc.

FACTOR INFLUENCING THE MICROBIOME BEFORE BIRTH

Current evidence does not support a major role for maternaldietary restrictions during pregnancy or lactation (1).However, there is some evidence that manipulation of thematernal gastrointestinal microbiota during pregnancymay have a health benet for the offspring. A group of256 women was randomized during the rst trimester ofpregnancy (2). Intensive dietary counseling providedby a nutritionist was done in the intervention group, withadditional randomization to receive probiotics (L. rhamnosusGG and B. animalisssp. lactisBb12) or not. The third groupwas a control group. The probiotic intervention resulted in

a decreased incidence of gestational diabetes mellitus (13percent versus 36 percent (diet/placebo) and 34 percent(control); P = 0.03) (2). Duration of pregnancy was normaland there were no adverse events in mothers in mothersor infants. The dietary counseling resulted in an increasedbirth weight (2). Probiotics given during the last trimesterof pregnancy and to mothers during lactation or to infantsif formula fed seem to reduced atopic dermatitis (3, 4).However, when the probiotics are started after birth, theoutcome is most of the time negative (5, 6).Prenatal exposure to proton pump inhibitors (PPI) and/orH2receptor antagonists (H2RAs)As taken during pregnancypredispose the offspring for allergic disease: the incidence ofatopic dermatitis (RR 1.32 (95 percent CI 1.06-1.64)), asthma

(RR 1.57 (95 percent CI 1.20-2.05)) and allergic rhinitis (RR 2.40(95 percent CI 1.42-4.04))(7).

BIRTH

The way of delivery, vaginally versus caesarean section (C.section) does inuence colonization of the gastro-intestinaltract. The gastro-intestinal (GI) microbiota of a baby bornthrough C. sections is very similar to the skin microbiota of themother, whereas after vaginal delivery, the GI microbiotais very similar to the vaginal colonisation (8). Escherichia-Shigellaand Bacteroidesspecies are underrepresented ininfants born by C. delivery. Infants born by elective C. deliveryhave a particularly low bacterial richness and diversity (9).

PRE-PROBIOTICS

YVAN VANDENPLAS

Vrije Universiteit Brussel, Department of Pediatrics, UZ Brussel, 1090 Brussels, Belgium


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transplantation is becoming more and more accepted as aneffective treatment of recurrent C. difciledisease (24). Therst reports are published on successful fecal transplantationin inammatory bowel disease, both in colitis ulcerosa andCrohns disease (25, 26). It is becoming obvious that the GImicrobiota may play a determinant role in many, if not all,auto-immune driven diseases since a relation has also beenreported with coeliac diseases and spondylarthritis (27, 28).Finally, the role of microbiome in central nervous systemdisorders is more and more investigated in depth (29). Therole that enteric short-chain fatty acids, particularly propionic(also called propanoic) acid, produced from ASD-associatedGI bacteria, may play in the etiology of some forms of autismspectrum disorders (30). Human populations that are partialmetabolizers of propionic acid are more common thanpreviously thought. The results from pre-clinical laboratorystudies show that propionic acid-treated rats display ASD-like repetitive, perseverative, and antisocial behavioursand seizure (30). Neurochemical changes, consistent andpredictive with ndings in autism spectrum disorder patients,including neuroinammation, increased oxidative stress,mitochondrial dysfunction, glutathione depletion, and altered

phospholipid/acylcarnitine proles, have been observed (30).The role of the environment on the development of atopicdisease is still heavily debated. It is not impossible that thepresence or absence of pets in a household exercise acertain effect on atopic disease through changes in theGI microbiota. Microbiota richness and diversity tendsto be increased in infants living with pets, whereas thesemeasures were decreased in infants with older siblings(31). Infants living with pets exhibited under-representationof Bidobacteriaceae and over-representation ofPeptostreptococcaceae; infants with older siblings exhibitedunder-representation of Peptostreptococcaceae (31).

FEEDING

Compared with breastfed infants, formula-fed infants havean increased richness of species, with overrepresentationof C. difcile(9). The differences in composition betweenbreast milk and formula are the major reason to explainthe difference in GI microbiota composition. Prebioticoligosaccharides, a well identied bidogenic factor, are thethird most important component in mothers milk, and theyare virtually absent in cow milk (32). It took some time beforeenough evidence was accumulated, but it has been shownthat mothers milk contains also small amounts of potentiallyprobiotic bacteria, which colonize the breast canaliculi (33).

During the last decades, the use of more sophisticated culture-dependent and -independent techniques, and the steadydevelopment of the -omic approaches are opening up thenew concept of the milk microbiome, a complex ecosystemwith a greater diversity than previously anticipated (33). Humanbreast milk is a source of diverse, active bacteria, includingbidobacteria, lactobacilli. Bacteria in human breast milkcolonize the gut of breast-fed infants (microbiota transferfrom the mother to the infant). Human breast milk containsnutritive factors supporting intestinal growth of bidobacteria.But many other differences in the composition of mothersmilk or formula contribute to the bifidogenic effect ofmothers milk: a low protein content, which is rich is wheyand alpha-lactalbumin, a high lactose content, and lowphosphor and low iron

Already 15 years ago, Grnlund et al showed that on thethird day of life, vaginally born infants have a much highercount of bidobacteria, lactobacilli and Bacteroidesthansthose born through C. section, while the number of clostridia iscomparable (10). But according to Salminen and coworkers,the number of clostridia is still different at the age of sevenyears, with vaginally born infants having higher counts forclostridia (11).According to the same authors, the difference in colonizationdisappears around the age of one month (10). Childrenborn after C. section have a signicant higher risk to developallergic disease such as allergic rhinoconjunctivitis andasthma (12). A meta-analysis clearly identied C. sectionas a risk factor to develop asthma (13). C. section is alsoassociated with other auto-immune diseases, such asdiabetes type 1 (14). Infants born by caesarean section(147/865, 17 percent) had a greater risk of diarrhoea (OR(adj)1.46, 95 percent CI 1.022 to 2.10) and sensitisation to foodallergens, both in adjusted (OR(adj) 2.06, 95 percent CI 1.123to 3.80) and stratied analyses (by cord blood IgE) (15).Caesarean delivery was not associated with colicky pain andatopic dermatitis (15). C. section was also identied as a risk

factor for obesity, even in adults (13).

AFTER BIRTH

The fecal microbiota among infants is characterized bya high variability. The proles are generally dominated byActinobacteria (mainly the genus Bidobacterium) andFirmicutes (with diverse representation from numerousgenera) (9). Differences in the neonatal gut microbiotaprecede the development of atopy, suggesting a crucialrole of the balance of indigenous intestinal bacteria for thematuration of human immunity to a nonatopic mode (16,17). At the age of 1 month, the presence of Echerichia coliand Clostridium difcileis associate with an increased riskto develop later in life eczema, recurrent wheeze, atopicdermatitis and allergic sensitization (16). However, a recentmeta-analysis reported to have found no evidence tosupport a protective association between perinatal useof probiotics and doctor diagnosed asthma or childhoodwheeze (18). Randomized controlled trials to date have notyielded sufcient evidence to recommend probiotics for theprimary prevention of these disorders (18). Of course, everyinfection, also a viral gastroenteritis disrupts the equilibrium ofthe GI microbiota (19). Bidobacteriumcolonization is lowerin patients with type 1 diabetes compared to the controlgroup, whereas Candida albicansand Enterobacteriaceae

other than E. colicolonization were increased (20). Highintestinal Bacteroides fragilisand low Staphylococcusconcentrations in infants between the age of 3 weeks and1 year are associated with a higher risk of obesity later in life(21). Experimental data in animals, but also observationalstudies in obese patients, suggest that the composition ofthe gut microbiota differs in obese v. lean individuals, indiabetic v. non-diabetic patients or in patients presentingother diseases associated with obesity or nutritionaldisbalance, such as non-alcoholic steatohepatitis (22).Microbial studies have elaborated the normal compositionof the gut microbiome and its perturbations in the setting ofinammatory bowel disease (23). This altered microbiomeor dysbiosis is a key player in the protracted course ofinammation in inammatory bowel disease (23). Fecal


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the GI microbiota to a pathogenic ora with yeast (48). Smallbowel bacterial overgrowth occurs in up to 25 percent ofthe children on proton pump inhibitors (49). The AmericanAcademy of Pediatrics reported that PPI are known to favourthe development of C. difcile(50). The lack of gastric acidityinduced by PPI is also a risk factor for the development ofpathogens in the respiratory tract, since broncho-alveolarlavage culture is more frequently positive in patients on PPI(51). As mentioned before, prenatal exposure to PPI increasesthe risk of the offspring to develop allergic disease (7).

CONCLUSION

The composition of the GI microbiota is inuence by manyexternal factors such as feeding and medication. A healthy,balanced microbiota is important for general health. Many,is not all, immune mediated diseases can be related to adysbiosis. Whether this regards an association, a cause ora consequence needs further evaluation. Future researchshould focus on the question if manipulation of the GImicrobiota can induce a better healing or control of auto-

immune diseases.

REFERENCES AND NOTES

1. Greer FR, Sicherer SH, Burks AW. American Academy ofPediatrics. Committee on Nutrition; AAP Section on Allergyand Immunology. Effects of early nutritional interventions onthe development of atopic disease in infants and children:the role of maternal dietary restriction, breastfeeding, timingof introduction of complementary foods, and hydrolyzedformulas. Pediatrics 2008;121:183-91.

2. Luoto R, Laitinen K, Nermes M, et al. Impact of maternalprobiotic-supplemented dietary counseling on pregnancyoutcome and prenatal and postnatal growth: a double

blind, placebo controlled. Br J Nutr. 2010;103:1792-9.3. Kalliomki M, Salminen S, Poussa T, et al. Probiotics during the

first 7 years of life: a cumulative risk reduction of eczema in arandomized, placebo-controlled trial. J Allergy Clin Immunol.2007;119:1019-21.

4. Niers L, Martn R, Rijkers G, et al. The effects of selectedprobiotic strains on the development of eczema (the PandAstudy). Allergy 2009;64:1349-58.

5. Soh SE1, Aw M, Gerez I, et al. Probiotic supplementation inthe first 6 months of life in at risk Asian infants--effects oneczema and atopic sensitization at the age of 1 year. ClinExp Allergy. 2009;39:571-8.

6. Taylor AL, Dunstan JA, Prescott SL. Probiotic supplementationfor the first 6 months of life fails to reduce the risk of atopicdermatitis and increases the risk of allergen sensitization inhigh-risk children: a randomized controlled trial. J Allergy ClinImmunol. 2007;119:184-91.

7. Mulder B, Schuiling-Veninga CC, Bos HJ, et al. Prenatalexposure to acid-suppressive drugs and the risk of allergicdiseases in the offspring: a cohort study. Clin Exp Allergy.2014;44:261-9.

8. Johnson CL, Versalovic J. The human microbiome and itspotential importance to pediatrics. Pediatrics 2012;129:950-60.

9. Azad MB, Konya T, Maughan H, et al. CHILD StudyInvestigators. Gut microbiota of healthy Canadian infants:profiles by mode of delivery and infant diet at 4 months.CMAJ 2013;185:385-94.

10. Grnlund MM, Lehtonen OP, Eerola E, et al. Fecal microflorain healthy infants born by different methods of delivery:permanent changes in intestinal flora after cesarean

contents. Phosphorus increases the buffering capacity ofthe stools and as a consequence it lowers the number ofbidobacteria. Breast milk has a high lactose content. Themetabolisation of lactose results in the production of acid,what induces a low buffering capacity of the stools, a lowfecal pH and thus an increase of bidobacateria. Therefore,formula fed infants have a different GI microbiota thanbreastfed infants. In infants fed classic standard infantformula, the GI microbiota is similar to that in adults (34). Theaddition of prebiotics oligosaccharides to infant formularesults in a change in GI microbiota composition, resultingin a similar microbiota in formula and breast fed infants (35).However, prebiotic oligosaccharides are not generic. Not alloligosaccharides have a similar effect. A new prebiotic blendof polydextrose and galacto-oligosaccharides may have asmaller bidogenic effect in young infants than a mixture ofgalactose- and fructo-olgosaccharides (GOS/FOS) (36). Thecounts of bidobacteria in the infants fed the polydextrose/galactose mixture were closer to the breast-fed group, butonly tended to be higher than control for total bidobacteria(P = 0.069) and Bidobacterium longum(P = 0.057) at 30 days(36). The same GOS/FOS mixture did result in higher levels

of sIgA in the stools compares to standard infant formula(37), while this could not be conrmed with the poydextrosemixture (36). A comparison between the physiological andbidogenic effects of different prebiotic supplements in infantformulae clearly demonstrates that prebiotic mixtures arespecic (38). Supplementation of an infant formula enrichedwith FOS at either 2.0 or 3.0 g/L did not appear to have anysignicant prebiotic effect with respect to bidobacteria,lactobacilli, C. difcile, E. colior Bacteroides(39). Anotherstudy with the specic GOS/FOS mixture showed that thedifferences in GI microbiota between the groups weremaintained during the second half of the rst year, even ifthe prebiotic supplementation was stopped at the age of 6months (40). It is well know from probiotics that they disappearfrom the GI microbiota between 1 and 2 weeks after stoppingthe administration. A prebiotic mixture and LCUPFAs given totoddlers reduces the risk to develop infections (41). Althoughof interest, the number of studies with symbiotic mixtures(pre- and probiotics) is relatively limited. Sazawal showedinteresting effects of a mixture with Bidobacterium lactisHN019 and GOS 2.5 g/100 g in 634 healthy children aged1- 3 years, for 12 months, showing a decreased incidence ofdysentery, days with severe illness days, days with fever, earinfections, iron decient anaemia, and as a result a bettergrowth and weight gain (42, 43).Micronutrients such as Vitamin D are reported to also regulatethe gut microbiome and to protect mice from dextran sodium

sulphate-induced colitis (44).

MEDICATION

It is well known, and logic, that the administration ofantibiotics changes the GI microbiota composition. Antibioticassociated diarrhea (AAD) is a frequent adverse event ofadministration of antibiotics, occurring in 10 to 20 percent (45).Among many other factors, the type of antibiotic given, thedosage, duration and the age of the patient are inuencingthe incidence of AAD. According to a review, the incidenceof AAD varies between 6 and 80 percent (46). However, moststudies report an incidence of 10 to 20 (47). Also acid-blockingmedications such as H2receptor antagonists and PPI, change


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body mass index during the first three years of life: anobservational study. Gut Pathog. 2011;3:8.

22. Delzenne NM, Neyrinck AM, Cani PD. Gut microbiota andmetabolic disorders: How prebiotic can work? Br J Nutr.2013;109 Suppl 2:S81-5.

23. Hold GL, Smith M, Grange C, et al. Role of the gut microbiotain inflammatory bowel disease pathogenesis: What have welearnt in the past 10 years? World J Gastroenterol.2014;20:1192-1210.

24. Jorup-Rnstrm C, Hkanson A, Sandell S, et al. Fecaltransplant against relapsing Clostridium difficile-associateddiarrhea in 32 patients. Scand J Gastroenterol. 2012;47:548-52.

25. Vandenplas Y, Veereman G, van der Werff Ten Bosch J, etal. Fecal microbial transplantation in a one-year-old girl withearly onset colitis - caution advised. J Pediatr GastroenterolNutr. 2014.

26. Kao D, Hotte N, Gillevet P, et al. Fecal microbiotatransplantation inducing remission in Crohns colitis and theassociated changes in fecal microbial profile. J ClinGastroenterol. 2014.

27. Stoll ML, Kumar R, Morrow C, et al. Dysbiosis of the entericmicrobiota in pediatric spondyloarthritis. Arthritis Rheumatol.2014.

28. De Palma G, Nadal I, Medina M, et al. Intestinal dysbiosisand reduced immunoglobulin-coated bacteria associatedwith coeliac disease in children. BMC Microbiol. 2010;10:63.

29. Macfabe D. Autism: metabolism, mitochondria, and themicrobiome. Glob Adv Health Med. 2013;2:52-66.

30. Wang Y, Kasper LH. The role of microbiome in central nervoussystem disorders. Brain Behav Immun. 2013.

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delivery. J Ped Gastroenterol Nutr 1999;28:19-25.11. Salminen S, Gibson GR, McCartney AL, Isolauri E. Influence of

mode of delivery on gut microbiota composition in sevenyear old children. Gut. 2004;53:1388-9.

12. Renz-Polster H, David MR, Buist AS, et al. Caesarean sectiondelivery and the risk of allergic disorders in childhood ClinExp Allergy 2005;35:1466-72.

13. Thavagnanam S, Fleming J, Bromley A, et al. A meta-analysisof the association between Caesarean section andchildhood asthma. Clin Exp Allergy 2008;38:629-33.

14. Cardwell CR, Stene LC, Joner G, et al. Caesarean section isassociated with an increased risk of childhood-onset type 1diabetes mellitus: a meta-analysis of observational studies.Diabetologia 2008;51:726-35.

15. Laubereau B, Filipiak-Pittroff B, von Berg A, et al. GINI StudyGroup. Caesarean section and gastrointestinal symptoms,atopic dermatitis, and sensitisation during the first year of life.Arch Dis Child 2004;89:993-7.

16. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiotacomposition and development of atopic manifestations ininfancy: the KOALA Birth Cohort Study. Gut 2007;56:661-7.

17. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiotacomposition and development of atopic manifestations ininfancy: the KOALA Birth Cohort Study. Gut 2007;56:661-7.

18. Azad MB, Coneys JG, Kozyrskyj AL, et al. Probioticsupplementation during pregnancy or infancy for theprevention of asthma and wheeze: systematic review andmeta-analysis. BMJ. 2013;347:f6471.

19. Nelson AM, Walk ST, Taube S, et al. Disruption of the humangut microbiota following Norovirus infection. PLoS One.2012;7:e48224.

20. Soyucen E, Gulcan A, Aktuglu-Zeybek AC, et al., Differencesin the gut microbiota of healthy children and those with type1 diabetes. Pediatr Int. 2013.

21. Vael C, Verhulst SL, Nelen V, et al Intestinal microflora and


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KEYWORDS: microbiome, microbiota, probiotics, prebiotics, human health, regulatory status, strategy of treatment

AbstractIt is well established that human microbiota and a balanced diet influence health of the individual.The microbiota has a physiological role, and its modification, e.g. by unbalanced nutrition or use ofantibiotics, can cause colonization by non-resident microorganisms, leading to different diseases. According to recentstudies, the correlation between microorganisms and humans has to be considered as a mutualistic-symbiotic relationshipand not a merely commensal activity.High-resolution spatial, temporal and functional microarrays of the human microbiota are still needed, and the effects ofenvironmental perturbations, the change in diet habits, and the today use of probiotics/prebiotics has to be deeper elucidated.Hence the necessity to further international rules to coordinate the production and distribution on the market of prebiotics andprobiotics, determining their composition, labels and advertisements for obtaining a sure safeness and effectiveness of this newcategory of special foods. This is the challenge for our future.

Probiotics and Prebioticsto save human microbiota enhancing health and well-being.Note I

INTRODUCTION

Human body is thesocial network where trillions ofmicroorganisms living on the skin, saliva, oral mucosa,gastrointestinal tract and genital area, participate inmaintaining the health (1, 2). Most of these microbialinhabitants, classified into 3 major phyla and referred to as themicrobiome/microbiota,perform tasks that appear not-harmful at all, but rather assist in maintaining the keyphysiological processes necessary for our wellbeing (Figure 1).It has been calculated that a human adult houses about 1012colony forming units (CFU) species of bacteria on the skin, 1010in the mouth, and 1014in the gastrointestinal tract. The latter

number is far in excess of the number of eukaryotic cells in allthe tissues and organs which comprise the human body (3, 4).These microorganisms are more than 10 times the human cellsaccounting for 1-3 precent total body mass. The 3.3 million ofgenes of the gastrointestinal tract (GI) result about 150 timesmore than the global human genome (5, 6).The purpose of the article is to share some informationsconnecting the human microbiota with health and regularuse of probiotics.

THE MICROBIAL ECOLOGY

The normal human microbiota consists of more than 1000species that are influenced by various factors, including

genetics, age, sex, stress, nutrition and diet. Despite theirrole is part of normal healthy physiology, once growbeyond their typical ranges, or populate atypical areas ofthe body, they cause a diseases and/or even simplymodify the GI biochemical environment.The Bacteroides thetaiotaomicron canbe a peculiarexample, since it can convert enzymatically thecarbohydrates vegetal-derived into glucose (6). Humangenome, in fact, does not possess these enzymes, so that

PRE-PROBIOTICS

PIERFRANCESCO MORGANTI1*, UMBERTO CORNELLI2, GIANCARLO GAZZANIGA3

*Corresponding author

1. 2nd University of Napoli, Dermatology Institute, Italy

2. Loyola University Medical School, Chicago, USA

3. ISCD Consultant, Via Innocenzo XI, 41, 00195 Roma, Italy

Figure 1.Microbiota/microbiome on human skinbody classified in 4 phyla.


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the self-created DNA and cathelicidin peptides causeauto-inflammation of the skin while, at the oppositecathelicidin suppression was shown in atopic dermatitis.In case of eczema and psoriasis an imbalance of skincommensals and potentially harmful microbes can takeplace influencing the immune system (20).Emerging evidences indicate that commensal microbesinhabiting skin, airway, and gut, protect these structuresagainst inflammatory disorders, despite little is knownabout determinants of their microbiota.A group of study from Helsinki University has shown howthe decline of biodiversity may contribute to the rise ofasthma, allergies and other chronic inflammatory diseasesamong people living in cities worldwide (21). Subjectsliving on farms or near forests had more diverse bacteriaon their skin and lower allergen sensitivity than individualsliving in areas with less environmental biodiversity, such asurban areas or near bodies of water. Furthermore, in thisstudy allergen- sensitive individuals have shown diversityon their skin of gamma-proteobacteria compared tohealthy subjects. The presence of gamma-proteobacteria,was associated with the expression of IL-10 in the blood of

the healthy subjects indicating its role in immunetolerance (21).These findings suggest how the increasing prevalence ofinflammatory diseases may be associated with the

changing biodiversity of both environment and

commensal skin bacteria. Moreover, ecological statisticalapproaches have revealed previously unrecognizedorganizations in architecture of the body complexmicrobial communities.

GASTROINTESTINAL MICROBIOTA

The gi microbiota starts in the mouth with a viable countof 108-1010 cfu of bacteria /g saliva, which reduces instomach 103, duodenum and jejunum102-109, increasesagain in ileum and colon to about 1010-1012respectively cfu/g (16). The large intestine is, there fore,the most diversely colonized and metabolically activeorgan in the human body,where colonic environment is

B. thetaiotaomicroninterference allows obtaining nutrientsfrom fruits and vegetables.It is becoming increasingly clear that the effect of themicrobial ecology in the body goes beyond the immunesystem, being implicated in immune-related disorders,such as diabetes, inflamm-aging, or obesity. Blaser et al.(7) have shown that Helicobacter pylori,the pathogenicbacterium causing gastric ulcers, is one of the normalcommensal of human stomach regulating both the levelof acidity and the production of ghrelin. This hormonestimulates the brain centre of appetite in competitionwith the appetite inhibitor leptin. The excess of acidity forthe disequilibrium of H. pyloriprovokes inflammation, andgastric ulcer, but its elimination with antibiotics stimulatesthe sensation of appetite and may cause obesity boundto the increase of ghrelin productionAccording to Lee and other authors, Bacteroides fragiliscontributes to maintain in equilibrium the immune system(8, 9), since it stimulates the production of both T-cellsregulators, and other pro-inflammatory T-cells throughthe polysaccharide -A positioned on its surface. Theequilibrium between these two kinds of T-cells reduces

the inflammatory process, avoiding damages to theimmune system.Unfortunately, due to the different way of living, in thelast century both B. fragilis and H. pyloriare disappearing(together with other anti-inflammatory commensalmicroorganisms) causing a decrease of the T-cellregulators. This could be the reason of the notablyincrease of the auto-immune diseases, such as type Idiabetes, atopic dermatitis, and multiple sclerosis, allconnected with genetic and environmental componentsunder regulation by the human microbiota.Research suggests that the relationship betweenmicroorganisms and humans is not merely commensal,but rather a mutualistic symbiotic one (9-12). On onehand, the microbiota of the human GI has been shown toproduce vitamins, thus contributing to nutrition, digestion,protection from establishment by alien microbes, andstimulation of the immunological tissues (12-14).Disorder and impairment of microbiota communities areassociated with conditions such as obesity, inflammatorybowel disease, and other critical illness (15-17). Thealteration of microbiota due to poor nutrition orantibiotics misuse can cause shifts in populations andcolonization by non residents microorganisms, leading togastrointestinal diseases.

SKIN MICROORGANISM

The skin is host of numerous bacterial and fungal species,either commensals or mutualistic, including transientpathogenic (18). The beneficial bacteria preventcolonization of pathogenic microorganisms, competingfor nutrients, secreting chemicals or stimulating the skinsimmune system (19).The skin is the first line of defence against infection, and aphysical and immunologic barrier (Figure 2) thatrecognizes beneficial and pathogenic microbes. Itproduces antimicrobial peptides, such as cathelicidins,that control the proliferation of skin microbiota andregulates cytokines inducing inflammation, angiogenesis,and skin re-epitheliziation (20). In psoriasis and rosacea,

Figure 2.Microbiome, antimicrobial peptides and skin immunology.


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PROBIOTIC AND PREBIOTIC

Probioticsare living non-pathogenic microorganisms that may

confer health benefits on the host (35, 36), while prebiotics aregenerally defined as nondigestible food ingredients thatbeneficially affect the host by selectively stimulating thegrowth and/or activity of one or a limited number of bacterialspecies already established in the colon, and thus in effectimprove host health (37, 38). Mechanisms of probiotic activityinclude (a) adhesion to the intestinal-lumen interface; (b)competition with pathogens for receptor binding, nutrientsand colonization;(c) enhancement of mucosal barrierfunction; (d) promotion of innate immune response; (e)production of bacteriocins; (f) and modulation of cell kinetics,with further mechanisms of action likely to be identified (36).On the other hand prebiotics are non-digestible andselectively fermented ingredients that allow specific changes,both in the composition and/or activity in the gastrointestinalmicroflora, conferring benefits(38, 39).Although all prebiotics are fibres, not all fibres are prebiotics.Thus, some known prebiotics, as inulin, are low digestiblecarbohydrates, some times associated with impairedgastrointestinal tolerance when consumed in large quantity,while other prebiotic fibres, such as wheat dextrin andpolydextrose, exhibit high gastrointestinal tolerability (40-43).Generally, probiotics, playing an important role in barrierdefence, may modulate the activity of many cells of theimmune system, including innate system cytokines,macrophages, epithelial cells and granulocytes, as well asadaptive system (33, 34). In any way probiotic bacterial

strains, having an almost exclusive saccharolytic metabolism,typical for lactobacilliand bifidobacteria, can be consideredpotentially beneficial exerting immune-activation, -deviationor -regulation/suppression responses. Thus, probiotic andprebiotic can significantly influence human health through arange of effects which include: (a)detoxification of

xenobiotics (42); (b) biosynthesis of vitamin K (43); (c)metabolic effects of fermentation of indigestible fibre (44); (d)positive influence on transit of luminal contents by peristalsis(45); (e) competition with pathogenic microbes for nutrientsand binding sites on mucosal epithelial cells (46); and (f)modulation of the hosts immune response (47). In any way,according to the joint FAO/WHO expert consultation,probiotics have been redefined as Live microorganismswhich when administered in adequate amounts confer a

favourable for bacterial growth due to its slow transittime, readily available nutrients ,and favourable ph.together with the gut immune system ,the mucosalmicroflora contributes significantly to the barrier thatprevents pathogenic bacteria invasion. The gimicrobiota ecosystem may be disturbed ,in fact, bypathogenic agents leading to a decreased bacterialdiversity and/or an unusual overgrowth of opportunisticpathogenic residential bacteria (23, 24). Recognition ofcommensal or pathogenic bacteria is of greatimportance to the mucosal immune system in elicitingpositive immune activation or negative re sponse. Inany way, a number of factors influence thecomposition of microbiota, such as the physiologicalcondition of the host ( age, stress, healthy status, etc.),composition of the diet and e nvironmentalcircumstances (antibiotic or antiseptic therapies,personal hygiene, etc.) (23, 24). At this purpose the useof prebiotic fibers and diet rich in plant-based foodseem to have health-promoting property ,beneficiallyaltering the microbial community (25). On the otherhand, probiotics show the ability to adhere in the

surface of digestive system and persisting longer in theintestinal tract ,stabilize the intestinal mucosal barrierand provide competitive exclusion of pathogenbacteria (26).

PRESENT RESEARCH-STUDIES ON MICROBIOTA

A high-resolution spatial-temporal and functionalmicroarrays of the global human microbiota, isnecessary to elucidate in a deeper way the effects ofenvironmental perturbations, such as the use ofantimicrobials and antibiotics, the changes in diethabitudes, and the today use of probiotics/prebiotics.Probably by the Second Human Microbe Project can bea way to improve the understanding the organization ofthe human microbiota system (27-29).This project, launched to survey microbial contentacross 242 healthy adults by a metagenomic approach,has developed a reference catalogue of microbialgenome sequences aimed to understand how thespecific habitats in the gut, genitourinary system, andskin may contribute to healthy and diseased state (28,29). Thus metagenomic studies using 16S rRNAsequencing in adults show that the vast majority of skinbacteria and gut flora fall into 4 major phyla (Figure 1)(22,30). Advances in DNA amplification and sequencing

technology has given the possibility to bypass thenormal culture steps, allowing more complete views ofhuman microbiota and their genetic content,collectively called microbiome. Defining in this way thenormal microbiological population on skin healthy sites(by metagenomics of skin microbiota), it will be possibleto provide a framework for investigating bacteria andfungi in different skin conditions, underlining newperspective to pathogenic factors and new therapeutictargets (Figure 3) (22,31).For all these reasons, many therapeutic strategiesaiming to restore a normal microbiota status by theselective use of probiotics and prebiotics should beuseful to rebalance the disordered microbial communityinfluenced by diseases or stress factors (17,32-34).

Figure 3.Metagenomics of skin microbiome.


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jun2012/nhgri-13. htm. (2012).6. Bijursell M.K., Martens E.C., et al., Functional genomic and

metabolic studies of the adaptation of a prominent adult humangut symbiot bacteroides thetaiotaomicron to the sucking period.

J Biol Chem, 281(47), 36269-79, (2006).7. Blaser M.j., Who Are We? Indigenous Microbes and The Ecology of

Human Diseases. EMBO Reports, 7(10), 956-960, (2006).8. Lee Y.K. and Mazmanian S.K., Has the Microbiota Played a Critical

Role in the Evolution of the Adaptive Immune System?. Science,330, 1768-1773, (2010).

9. Guarner F., Malagelada J.R., Gut flora in health and disease.Lancet, 361(9356), 512-519, (2003).

10. Steinoff U., Who controls the crowd? New findings and oldquestions about the intestinal microflora. Immunology letters,99(1), 12-16. (2005).

11. OHara A.M., Shanahan F., The gut flora as a forgotten organ.EMBO Reports, 7(7), 688-693, (2006).

12. Hand T.W., Dos Santos L.M., et al., Acute gastrointestinal infectioninduces long-lived microbiota-specific T cell responses. Science,337,1553-1556, (2012).

13. Rudin A., Lundell A.C., Infant B cell memory and gut bacterialcolonization. Gut Microbes, 3, 474-475, (2012).

14. Patil D.P., Dhotre D.P., et al., Molecular analysis of gut microbiotain obesity among Indian individuals.J Biosci, 37, 647-657, (2012).

15. Manichanh C., Borruel N., et al., The gut microbiota in IBD. NatRev Gastrienterol Hepatol, 9, 599-608, (2012).16. Hakansson A. and Molin G., Gut Microbiota and Inflammation.

Nutrients, 3, 637-682 (2011).17. Grice E.A., Kong H.H., et al., A diversity profile of the human skin

microbiota. Genome Res, 18, 1043-1050, (2008).18. Ursel L.K., Clemente J.C., et al., The interpersonal and

intrapersonal diversity of human-associated microbiota in keybody sites.J Allergy Clin Immunol, 129, 1204-1208, (2012).

19. Schauber J. and Gallo R.L., Antimicrobial peptides and the skinimmune defence system.J Allergy Clin Immunol, 122(2), 261-2661,(2008).

20. Naik S. et al., Compartmentalized control of skin immunity byresident commensals. Protective role of skin microbiota.ScienceDaily. Retrieved July 20, 2013. www.sciencedaily.con/

release/2012/07/120726153947.htm. (2012).21. University of Helsinki., Biodiversity loss may cause increase in

allergies and asthma. ScienceDaily. Retrieved, July 20, 2013, fromwww. sciencedaily. com/release/2012/05/120507154114.htm.(2012, may 7).

22. Chen Y.E. and Tsao H., The skin microbiome: Current perspectiveand future challenges.J Am Acad Dermatol, 69,143-155, (2013).

23. Slavin J., Fiber and Prebiotics: Mechanisms and Health Benefits.Nutrients, 5, 1417-1435, (2013).

24. De Filippo F.C., Cavallieri D., et al., Impact of diet in shaping gutmicrobiota revealed by a comparative study in children fromEurope and rural Africa. Proc Natl Acad Sci USA,107, 14691-14696(2010).

25. Brownawell A.M., Caers W., et al., Prebiotics and the healthbenefits of fiber: Current regulatory status, future research, and

goals.J Nutr , 142, 1-13 (2012).26. Turnbaugh P.J., Ley R.E., et al., The human microbiome project.

Nature, 449, 804-810, (2007).27. Peterson J., Garges S., et al., The NIH human microbiome project.

Genome Res, 19, 2317-2323, (2009).28. Human Microbiome Project Consortium. A framework for human

microbiome research. Nature, 486, 215-221, (2012).29. Human Microbiome Project Consortium. Structure, function and

diversity of the healthy human microbiome. Nature, 486, 207-214,(2012).

30. Gao Z., Tseng C.H., et al. Molecular analysis of human forearmsuperficial skin bacterial biota. Proc Natl Acad SciUSA, 104(8),

2927-32 (2007).

Readers interested in a full list of references are invited to visit ourwebsite at www.teknoscienze.com

health benefits on the host while prebiotics confer benefitsallowing specific changes in the composition and activity ofthe GI microflora (23). Thus, selection and combination ofboth probiotics and prebiotics seem to represent anefficacious approach to the control of some immunehypersensitivity/allergy reactions, acting also as regimen forlong health and well-being. The experts agreed, in fact, thatadequate scientific evidence exists to indicate [...] healthbenefits from consuming food containing probiotics, [...]specific strains of probiotics are safe and able to confer somehealth benefits on the host; and these benefits includecondition such as gastrointestinal infections, certain boweldisorders, allergy, urogenital infection, also if theirapplication to prevent and treat these disorders should bemore widely considered by the medical community. Inaddition, there is emerging evidence to indicate thatprobiotics can be taken by healthy people to prevent certaindiseases and modulate host immunity and supporting also agut-microbiome brain connection may be of help in humanneurodevelopment disorders (49).

RESEARCH PERSPECTIVES AND REGULATORY STATUS

In conclusion prebiotics have essentially the same aim asprobiotics, which is to improve host health via modulation ofthe intestinal flora, although by a different mechanism (48).However, on one hand determining the precise compositionof secreted products from probiotic bacteria is challengingbecause of its dependence from species, strain, microenvironment, culture conditions and their adhesivity onhuman epithelium. On the other hand, prebiotics exhibit adiverse range of physiological effects depending on theirphysico-chemical characteristics, such as solubility,fermentability, and viscosity.Moreover, there is the necessity to further rules because theregulatory status of probiotics and prebiotics as a componentin food is currently not established on an international basis.In addition, even if these products are consideredsafe, thereis a need for refinement of in vitro and in vivotests to betterpredict their function in humans. This goal will be addressedby the application of good manufacturing practices and thedefinition of the shelf-life conditions reported on the label,together with verifiable health claims. Finally, surveillancesystem will be necessary as well as trace-back and postmarketing surveillance to control the eventual side-effectsassociated with the consume of prebiotics and probiotics inthe long period of time (48). These are the future challengesto save the human microbiota enhancing well-being and the

quality of our life.


1. Grice E.A. and Segre J.A., The skin microbiome. Nat RevMicrobiology, 9, 244-253, (2013).

2. Sears C.L., A dynamic partnership: Celebrating out gut flora.Anaerobe, 11(5), 247-251, (2005).

3. Todar K., The Normal Bacterial Flora of Humans. In: Todar K TheGood, the Bad, and the Deadly. Todar Online Textbook ofBacteriology. www. textbookofbacteriology.net. (2013).

4. Grice E.A., Kong H.H., et al., Topographical and Temporal Diversityof the Human Skin Microbiome. Science, 324, 1190-1192. (2009).

5. MacDougall R., NIH Human Microbiome Project defines normalbacterial makeup of the body. www.nih. gov/news/health/



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Industry perspective

NOME COGNOME


indirizzo 1

indirizzo 2

15

KEYWORDS: breastfeeding, formula feeding, infant nutrition, arachidonic acid, docosahexaenoic acid

AbstractBreastfeeding is the most natural way to feed an infant. Moreover global experts recommend exclusivebreastfeeding for the first six months of life. DHA and ARA are, respectively, omega-3 and omega-6, long-chain polyunsaturated fatty acids. These important fatty acids are always present in breast milk. In many instances, however, there arecompelling reasons why breastfeeding or exclusive breastfeeding is not possible. The article reviews the numerous benefits reported forexclusive breastfeeding as well as other studies documenting the benefits of DHA- and ARA-supplemented infant formulas.

Enrichment of infant formula withdocosahexaenoic acid (DHA)

and arachidonic acid (ARA)

INTRODUCTION

Paediatricians and nutritionists recommend exclusivebreastfeeding of infants until 6 months of age. It is helpful fornew mothers to have current information regardingbreastfeeding as well as for formula feeding. This brief articlereviews recent key published literature addressing the benefitsof DHA and ARA when provided in breast milk or in the caseof those who cannot breast feed, in infant formula.

BREASTFEEDING

Breastfeeding is the gold standard and the natural way tofeed an infant. Paediatricians and nutritionists are unanimousin recommending that mothers exclusively breastfeed theirinfants for about six months. In its 2012 policy statement, theAmerican Academy of Paediatrics reaffirmed this advice,

adding that complementary foods can be introduced at 6months with the continuation of breastfeeding for one year orlonger as mutually desired by the mother and infant.(1) TheWorld Health Organization also recommends exclusivebreastfeeding for 6 months.(2)

Advantages

The advantages of exclusive breastfeeding for 3 months ormore include significantly lower r isks of respiratory tract andear infections, asthma, bronchiolitis, atopic dermatitis,gastroenteritis, sudden infant death syndrome and severalother diseases.(1) Breastfeeding also confers greaterprotection against infections because it contains manyimmunological and protective factors.(3, 4) Human milk alsopromotes the maturation of the infants immune system. Other

advantages of breastfeeding for 4 months or longer mayinclude higher scores for fine motor skills and communicationat age 3 compared with infants breastfed less than 4months(5) and higher academic achievement at 10 years ofage, especially in boys.(6) The American Academy ofPaediatrics noted that the health benefits of breastfeedingare greater when the infant is fed longer than 3 months.(1)

Evidence suggests that breastfeeding may have beneficialeffects on the long-term health of the offspring, though notall studies are conclusive. Breastfeeding has beenassociated with a lower risk of developing insulin-dependent (Type 1) diabetes,(7) metabolic syndrome,(8)non-insulin-dependent (Type 2) diabetes,(9) inflammatorybowel disease(10) and a slightly lower risk of heartdisease(11) and stroke,(12) although the risk of heartdisease and stroke has not been confirmed by others.(13)Children who were exposed to diabetes during fetal life

had less fat tissue if they were breastfed for at least 6months.(14) There is also evidence that children andadolescents who were breastfed for at least 6 months wereless likely to develop mental health problems.(15) The linksbetween breastfeeding and risks of allergic disease areinconsistent and vary with the type of allergy, family historyof allergic disease and other factors.(16) Environmentalexposures, changes in gene expression, maternal andinfant gene types, and various dietary factors have all beenlinked to the risk of allergy.(17-19)

Whether breastfeeding partly protects against thedevelopment of overweight and obesity later in life iscontroversial. Several studies have confirmed that rapidgrowth in early infancy and weight are(20) associated with

INFANT NUTRITION

SHEILA T. GAUTIER

DSM, 6480 Dobbin Road, Columbia, MD 21045, USA


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Agro FOOD Industry Hi Tech - vol 25(5) - September/October 201416 Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

(41) Many studies have demonstrated that infantsconsuming formula grow as well(48) as breastfed infants andhave comparable neurodevelopmental scores, withoutadverse effects.(42)

Mothers who do not breastfeed have many choices amongformulas. Brands differ by the source of protein(s) and thetypes of fat and carbohydrates they have. The main proteinsare soy, various cows milk proteins or hydrolyzed proteins,which have been broken down to increase their digestibility.Some formulas are free of the sugar lactose. Mostpaediatricians recommend a formula with added iron toprevent anaemia. Special formulas are available for preterminfants and those with medical needs. All infant formulasmarketing in the U.S. must meet federal nutrient requirementsand are regulated by the Food and Drug Administration.

Long-chain PUFAs

Since 2001, infant formula manufacturers have been permittedto add two long-chain polyunsaturated fatty acids to their

products. These are DHAand ARA. The amounts

added are similar to theworldwide averages ofthese fatty acids that arefound naturally in humanmilk. The addition of DHAand ARA ensures that theinfant receives sufficientamounts of these fattyacids for healthy brainstructure and functionand visual development.The only DHA used ininfant formula in the U.S.comes from a vegetarianand sustainable source,algae.

Visual and

Neurodevelopmental

Outcomes

There is evidenceshowing that term orpreterm infants

supplemented with DHA in infancy may have improvedvisual acuity, attention and cognitive performance inchildhood compared with infants fed unsupplementedformula, but results vary with the dose, time of assessment

after birth and for cognition in particular, the measurementtool(s). Term infants fed different amounts of DHA with ARAhad higher visual acuity scores at 12 months of agecompared with those consuming the unsupplementedformula.(43) The same study also reported thatsupplemented infants fed the lower doses of DHA with ARAspent longer processing an active stimulus thanunsupplemented infants.(44) In contrast to these reports, ameta-analysis of four large randomized trials concluded thatDHA and ARA supplementation of infant formula had noclinically meaningful effect on infant neurodevelopment at18 months as assessed with the Bayley Scales of InfantDevelopment.(45) Similarly, 1 percent DHA with ARAsupplementation of preterm infants born at less than 33weeks gestation was not associated with any effect on

being overweight and obese in childhood andadolescence.(21, 22) However, the evidence is inconclusivewhether breastfeeding protects against the laterdevelopment of being overweight and obese in theoffspring.(23-27) Breastfeeding is associated with a slowerrate of weight gain in infancy and lower plasma insulin levels,which are associated with decreased fat storage.(28) Fasterinfant growth and weight gain have been attributed to thehigher protein content of infant formula compared withbreast milk.(29) Breastfeeding or formula with lower proteincontent restored the growth rate to that observed inbreastfed infants.(30, 31) Others suggested that the higherenergy density and volume of formula consumed contributeto the faster growth rate of formula-fed infants.(32)

Long-chain PUFAs

Until the development of fatty-acid-supplemented infantformula, breastfeeding was the only way to ensure that aninfant received long-chain polyunsaturated fatty acids(PUFAs), including both DHA and ARA. Breast milk naturallycontains both of thesePUFAs, which are

necessary for braindevelopment andfunction,(33) visualfunction, immunesystem development(34)and other activities.DHA must be obtainedfrom the diet in apreformed statebecause the usualWestern diet providestoo little from conversionof its fatty acidprecursor alpha-linolenic acid or frombody stores.(35, 36)Mothers who eatseafood regularly ortake fish or algal oilsupplements havehigher levels of DHA intheir breast milk thanwomen who do nothave dietary sources of these fatty acids.(37) Women whodo not eat fish, vegetarians and vegans,(38) have lowerlevels of DHA in their breast milk compared with omnivorewomen or those who eat fish. Most, but not all, infant

formulas now contain added DHA and arachidonic acid(ARA), an omega-6 fatty acid that also helps support infantdevelopment.

INFANT FORMULA FEEDING

Mothers may choose not to breastfeed their infant becauseof medical conditions, work-related issues, personalpreferences, social and cultural perceptions, lack ofappropriate support and other circumstances,(39) but thereare few medical reasons not to breastfeed.(40) Mothers whodecide not to breastfeed, regardless of the reason, can bereassured that their infant will be healthy, well-nourished andhave normal scores for growth and cognitive development.

Credit for the photographs goes to (c) DSM Nutritional Products Ltd.


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from Epidemiological Studies, Proc Nutr Soc, 70 (4), 478-484 (2011).10. Klement, E., Cohen, R. V., Boxman, J., et al. Breastfeeding and Risk

of Inflammatory Bowel Disease: A Systematic Review with Meta-Analysis,Am J Clin Nutr, 80 (5), 1342-1352 (2004).

11. Singhal, A. The Early Origins of Atherosclerosis,Adv Exp Med Biol,646 51-58 (2009).

12. Rich-Edwards, J. W., Stampfer, M. J., Manson, J. E., et al.Breastfeeding During Infancy and the Risk of CardiovascularDisease in Adulthood, Epidemiology, 15 (5), 550-556 (2004).

13. Martin, R. M., Davey Smith, G., Mangtani, P., et al. Breastfeedingand Cardiovascular Mortality: The Boyd Orr Cohort and aSystematic Review with Meta-Analysis, Eur Heart J, 25 (9), 778-786(2004).

14. Crume, T. L., Ogden, L., Maligie, M., et al. Long-Term Impact ofNeonatal Breastfeeding on Childhood Adiposity and FatDistribution among Children Exposed to Diabetes in Utero,Diabetes Care, 34 (3), 641-645 (2011).

15. Oddy, W. H., Kendall, G. E., Li, J., et al. The Long-Term Effects ofBreastfeeding on Child and Adolescent Mental Health: APregnancy Cohort Study Followed for 14 Years,J Pediatr, 156 (4),568-574 (2010).

16. Jennings, S., Prescott, S. L. Early Dietary Exposures and FeedingPractices: Role in Pathogenesis and Prevention of AllergicDisease?, Postgrad Med J, 86 (1012), 94-99 (2010).

17. Karmaus, W., Dobai, A. L., Ogbuanu, I., et al. Long-Term Effects ofBreastfeeding, Maternal Smoking During Pregnancy, and RecurrentLower Respiratory Tract Infections on Asthma in Children,J

Asthma, 45 (8), 688-695 (2008).18. Jones, A. P., Tulic, M. K., Rueter, K., et al. Vitamin D and Allergic

Disease: Sunlight at the End of the Tunnel?, Nutrients, 4 (1), 13-28(2012).

19. West, C. E., Videky, D. J., Prescott, S. L. Role of Diet in theDevelopment of Immune Tolerance in the Context of AllergicDisease, Curr Opin Pediatr, 22 (5), 635-641 (2010).

20. Andersen, L. G., Holst, C., Michaelsen, K. F., et al. Weight andWeight Gain During Early Infancy Predict Childhood Obesity: ACase-Cohort Study, Int J Obes (Lond), (2012).

21. Monteiro, P. O., Victora, C. G. Rapid Growth in Infancy andChildhood and Obesity in Later Life--a Systematic Review, Obes

Rev, 6 (2), 143-154 (2005).22. Stettler, N., Zemel, B. S., Kumanyika, S., et al. Infant Weight Gain

and Childhood Overweight Status in a Multicenter, Cohort Study,Pediatrics, 109 (2), 194-199 (2002).

23. Beyerlein, A., von Kries, R. Breastfeeding and Body Composition inChildren: Will There Ever Be Conclusive Empirical Evidence for aProtective Effect against Overweight?,Am J Clin Nutr, 94 (6 Suppl),1772S-1775S (2011).

24. Shields, L., Mamun, A. A., OCallaghan, M., et al. Breastfeedingand Obesity at 21 Years: A Cohort Study,J Clin Nurs, 19 (11-12),1612-1617 (2010).

25. Kramer, M. S., Matush, L., Vanilovich, I., et al. Effects of Prolongedand Exclusive Breastfeeding on Child Height, Weight, Adiposity,and Blood Pressure at Age 6.5 Y: Evidence from a LargeRandomized Trial,Am J Clin Nutr, 86 (6), 1717-1721 (2007).

26. Neutzling, M. B., Hallal, P. R., Araujo, C. L., et al G. Infant Feedingand Obesity at 11 Years: Prospective Birth Cohort Study, Int JPediatr Obes, 4 (3), 143-149 (2009).

27. Michels, K. B., Willett, W. C., Graubard, B. I., et al. A LongitudinalStudy of Infant Feeding and Obesity Throughout Life Course, Int JObes (Lond), 31 (7), 1078-1085 (2007).

28. Oddy, W. H. Infant Feeding and Obesity Risk in the Child,Breastfeed Rev, 20 (2), 7-12 (2012).

29. Escribano, J., Luque, V., Ferre, N., et al. Effect of Protein Intake andWeight Gain Velocity on Body Fat Mass at 6 Months of Age: The EuChildhood Obesity Programme, Int J Obes (Lond), 36 (4), 548-553(2012).

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Readers interested in full list of references are invited to visit ourwebsite at www.teknoscienze.com

mental development scores on the Bayley Scales at 18months, but scores were significantly higher among the DHA-supplemented girls.(46) These authors also reported otherbenefits of high-dose DHA.

Cognitive Outcomes

A concern with global cognitive tests measured at a singletime point is their lack of sensitivity to the differentdevelopmental time courses for discrete cognitive functions.(47) Thus, different aspects of cognition mature at differentrates. A recent study showed that in infants whose scores didnot differ on the Bayley Scales of cognitive performance at 18months, exhibited positive effects of DHA and ARAsupplementation at 3 to 6 years of age when assessed with abattery of cognitive tests at 36, 42, 48, 60 and 72 months ofage.(48) The results suggested that the benefits of DHA andARA supplementation were observed in early measures ofattention, preschool measures of rule learning andimplementation and later measures of verbal ability. Thesefindings suggest that more sophisticated measures of cognitivedevelopment over longer time may yield more informationabout the effects of DHA and ARA on cognitive development.

SUMMARY

There are considerable benefits related to exclusivebreastfeeding during the first 6 months of an infants life.However in situations in which a mother is unable tobreastfeed her infant exclusively for 6 months, there arecurrently available commercial infant formulas containingthe long-chain polyunsaturated fatty acids, DHA and ARAfrom algal single cell oils which can provide similar benefitsto human milk. The information in this brief review may helpparents make an informed choice about their infantsnutrition and also help them to realize that breastfeedingversus formula feeding need not be mutually exclusive.


1. American Academy of Pediatrics. Breastfeeding and the Use ofHuman Milk, Pediatrics, 129 e827-e821 (2012).

2. Habicht, J. P. Expert Consultation on the Optimal Duration ofExclusive Breastfeeding: The Process, Recommendations, andChallenges for the Future,Adv Exp Med Biol, 554 79-87 (2004).

3. Chirico, G., Marzollo, R., Cortinovis, S., et al. Antiinfective Propertiesof Human Milk,J Nutr, 138 (9), 1801S-1806S (2008).

4. Newburg, D. S. Innate Immunity and Human Milk,J Nutr, 135 (5),1308-1312 (2005).

5. Oddy, W. H., Robinson, M., Kendall, G. E., et al. J. Breastfeedingand Early Child Development: A Prospective Cohort Study,ActaPaediatr, 100 (7), 992-999 (2011).

6. Oddy, W. H., Li, J., Whitehouse, A. J., et al. Breastfeeding Durationand Academic Achievement at 10 Years, Pediatrics, 127 (1), e137-145 (2011).

7. Patelarou, E., Girvalaki, C., Brokalaki, H., et al. Current Evidence onthe Associations of Breastfeeding, Infant Formula, and Cows MilkIntroduction with Type 1 Diabetes Mellitus: A Systematic Review,Nutr Rev, 70 (9), 509-519 (2012).

8. Khuc, K., Blanco, E., Burrows, R., et al. Adolescent MetabolicSyndrome Risk Is Increased with Higher Infancy Weight Gain andDecreased with Longer Breast Feeding, Int J Pediatr, 2012 478610(2012).

9. Owen, C. G., Whincup, P. H., Cook, D. G. Breast-Feeding andCardiovascular Risk Factors and Outcomes in Later Life: Evidence


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Industry perspective

NOME COGNOME


indirizzo 1

indirizzo 2

KEYWORDS: cookies quality, medicinal plants, functional food, sensory evaluation, colour, texture

AbstractVitalplant extract, composed of non-toxic medicinal plants to enhance metabolism, can be consideredfunctional food ingredient because of its biologically active constitutes, antioxidant and antimicrobialproperties. Sensory and instrumental properties of new cookie formulations, developed by supplementing the basic cookie formulawith 2 percent, 4 percent, and 6 percent Vitalplant extract were tested in this study. Referring to the results, the extract additioncaused statistically significant (P


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Cookie surface colour measurements

Colour values of cookies were determined by chromameterMINOLTA, CR-400 (Minolta Co., Ltd., Osaka, Japan). At fivemeasured points of top and bottom (centre and at thecorners of cookies), surface colour of cookies was recordedfor 10 randomly chosen cookies per batch and averaged.The results were expressed as lightness (L*), redness-greenness (a*), and yellowness-blueness (b*), according toCIE L*a*b* system.

Textural analysis

Textural analysis of cookies was conducted using a TA.XTPlusTexture Analyzer (Stable Micro Systems, England, UK),equipped with 3-point bending rig (HDP/3PB), and a 5 kg loadcell. Ten measurements per each cookies type were made.

Statistical analysis

Results were expressed as the mean of replications SD forall measurements. Analysis of variance (ANOVA) andDuncans multiple range tests was used to comparemeans at 5 percent significance level by using thestatistical data analysis software system XLSTAT, version

(2012.2.02) (http://www.xlstat.com/).

RESULTS AND DISCUSSION

Sensory quality of enriched cookies was defined in the termsof appearance, texture, odour and taste. The obtained resultsare shown in Table 1.

Because of the experts were highly familiar with this type ofproduct they evaluated the samples in their general conceptof the cookies quality defined based on evaluation of threeprimary sensory properties that determined the overall qualityof the cookies depending on the actual scores:unacceptable (4.5). Sensory evaluation showed that the cookieenriched with 4 percent extract had almost excellent overallquality (4.47)(Table 1). Scores for individual sensory properties,except for odour and taste of control sample, indicated verygood or excellent quality of all cookies. In addition to, dataanalysis of the odour and taste scores revealed thestatistically significant difference between the control sampleand all enriched cookies. The visual aspect of appearancewas evaluated in a similar way, comprehensive with differentlevels of quality. Although it was possible to easily differentiatebetween samples by colour due to different levels of theethanolic extract, overall perceived appearance by panelshowed that there was no statistical difference between thesamples in terms of appearance, taking into account thecomplexity of this property. Similar results were obtained for

wheat flour (13.3 percent water content, 0.39 percent db ashcontent, and 10.5 percent db protein content), obtainedfrom Zitko (Mill Baka Topola, Serbia) was used for theformulation of the cookies. Vegetable fat was obtained fromPuratos, Serbia. Salt, powdered sugar and baking powderwere purchased in a local food store.

Preparation of plant extracts for the formulation of cookiesAs explained elsewhere (2), crude plant extracts wereobtained by maceration with ethanol/water mixture (80:20,v/v), with the ratio of raw materials to ethanol solution of 1:10,for 24 h at room temperature and subsequently extracted inan ultrasonic bath at room temperature for 10 min. Afterfiltration through a filter paper (Whatman, Grade 4 Chr, UK)and vacuum-evaporation of the solvent at 40C, the extractswere stored at -4C until further use.

Preparation of cookies

Baking trials were conducted under laboratory conditions.Dough mixing, processing and baking were performed onlaboratory-scale equipment. The basic cookie doughformulation (control sample) contained 100 g of refined

wheat flour, 40 g of vegetable fat, 30 g of powdered sugar, 1g of salt, 1 g of baking powder. By adding the ethanolicextract (suspended in approximately 5 g of water) of herbalmixture Vitalplant to the basic formulation, cookies wereprepared to provide three variations: 2 percent, 4 percent,and 6 percent addition (supplementation with extractobtained from 2, 4, and 6 g of Vitalplant mixture,respectively). The amount of water added varied to obtaincohesive dough suitable for the production of mould-cut cookies. Ingredients were mixed in Diosna mixer asper all-in method. All ingredients were mixedtogether in one phase for 15 min. The formed doughwas packed in polyethylene bags and left to rest 0.5hours at ambient temperature (22-24C). Following therest time, the dough was sheeted to a final thickness,approximately 5 mm on a pastry break, and cut outusing a rectangular cutter. Dough pieces were bakedfor 12 min in a deck oven at 170C. After cooling, thecookies were placed in polyethylene bags and storedat ambient temperature until further examination (1).

Physical characteristics and sensory evaluation

Sensory evaluation was conducted by six experiencedpanellists (35-50 years old), 24 h after baking. Samples wereevaluated in two replications and presented separately in abalanced order. Sensory profiling was performed using ageneric descriptive analysis technique, which included

selected representative properties of cookies (11, 12), usinga 5-points method. Each mark was described with words,using previously prepared standard cards (12, 13, 14). Foreach sensory property, fixed importance coefficient and (IC)was standardized by the panel. The importance coefficientswere balanced in the way that their sum equals 4.0. Theobtained marks were multiplied by the following IC: forappearance (shape and surface) (IC = 0.8); for structureand break (IC = 0.8); for chewiness (IC = 0.8); for odour (IC =0.6); and for taste (IC = 1.0). The quantitative expression ofthe total product quality is obtained as the weighted meanvalue of the scores for each evaluated property. Qualitycategory was determined in dependence on scores:unacceptable (4.5).

Table 1.Sensory evaluation of cookies.Values are means SD of six panellists. Values with the different superscriptwithin a raw are statistically different (P < 0.05).Quality category wasdetermined in dependence on scores: unacceptable (4.5).


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structure and break, noting that the sample with the addition of 4percent extract, achieved the highest score (4.50). The addition of 2percent extract resulted in the significantly softer product comparedto the addition of 6 percent, while there was no significant differencein the remaining two samples. In addition, data analysis revealed astatistically significant difference between the scores for odour andtaste of all enriched cookies and control sample. The addition ofethanolic extract resulted in no significant differences betweenrelevant sensory properties of all enriched cookies, but cookies withhigher levels of enrichment were evaluated higher scores, in relationto odour in favour of 6 percent (4.55), and to taste in favour of 4percent (4.67) (Table 1). This is confirmed by the fact thatVitalplant mixture extract, applied at three levels ofsupplementation significantly improved the odour and the taste ofthe cookies. In general, the obtained results showed thatVitalplant mixture in the form of extract may be used in cookieformulations without producing a negative impact on the sensoryproperties.The surface colour of a baked product is, together with texture andflavour, a very important property for the acceptability of bakedgoods by consumers (15). The obtained colour values of cookiesamples are presented in Table 2.

According to Chevallier et al., (16) the colour of the cookies topsurface is under the influence of non-enzymatic browning during thebaking process, caused by the reaction between reducing sugars andamino acids, as well as by starch dextrinisation and sugarcaramelisation. Regarding colour, the specific additional componentswith which colour was associated in the cookies, contributed tosignificant differentiation of the enriched cookies to each other andrelative to the control sample.

The obtainedL* values of the top and bottom surface showed thatlightness decreased significantly (P< 0.05) , from 78.86 to 58.73 for topsurface, and from 72.23 to 55.12 for bottom surface, with the increase inthe level of supplementation. These results were in accordance withpreviously published statements of the other authors (17, 18, 19), whofound that the incorporation of different compound enrichments maycontribute to darker top surface. On the other hand, the incorporationof different amount of ethanolic extract contributed to increase in thepositive a*and b* values. There was a statistically significant increase(from 0.06 to 6.39) in the positive a* value of the top surface in allenriched cookies compared to the control sample. In addition, theamount of 4 and 6 percent enrichment led to the significant increase inthe red tonality compared to the control (+a = 0.50) and the samplewith 2 percent enrichment (+a = 6.84) in the case of determining thecolour of the bottom surface (Table 2). Furthermore, yellow tonality

Table 2.Colour values of cookiesValues are means SD of twenty-five measurements.Values with the different superscript within a column are statisticallydifferent (P < 0.05).



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bakery producers. The addition of the medicinal plant mixtureextract may be used in the cookie formulations withoutadversely affecting their sensory properties. Cookiessupplemented with 4 percent Vitalplant mixture extract wereestimated as the best sample. Further research needs to bedone in order to examine consumers acceptance of newproducts with appropriate substitutions relative to liking,especially in the terms of colour, but without neglecting thenecessary quantities that would contribute to health benefits.

CKNOWLEDGEMENTS

This paper is a result of the researches withinthe Project No. TR 31029 ( The Ministry ofEducation, Science and and TechnologicalDevelopment, Republic of Serbia) and theProject No. 114-451-4382 (the ProvincialSecretariat for Science and TechnologicalDevelopment, Autonomous Province ofVojvodina, Republic of Serbia)

REFERENCESAND NOTES

1. A. Mian, O. imurina et al., In Proceedings of the 9th

International Symposium on Interdisciplinary Regional Research,

Environmental, Health and Humanity ISSUES in the Down

Danubian Region: Multidisciplinary Approaches, pp. 165-171

(2009).

2. A. Mian, N. Mimica-Duki et al., Cent Eur J Chem, 9 (1), pp. 133-

142 (2011a).

3. A. Mian, N. Mimica-Duki et al.,J Food Sci,76(9), pp. 1239-1244

(2011b).

4. Lj. ari, I. abarkapa et al., Food Processing, Quality and Safety,

36 (1-2), pp. 1-6 (2009).

5. W. Verbeke, J. Scholderer et al.,Appetite, 52,pp. 684-692 (2009).

6. R. Krutulyte, K. G. Grunert et al., Food Qual Prefer, 22,pp. 11-16

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7. K. Glanz, M

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