Mycotoxicogenic fungal inhibition by innovative cheese cover with aromatic plants Armando Moro ...

225 Pages·2013·3.76 MB·English

UNIVERSIDAD DE CASTILLA-LA MANCHA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
Departamento de Ciencia y Tecnología Agroforestal y Genética

Doctoral Thesis

Alternatives to control microbiological cheese defects: Use of
aromatic plants

Celia Mª Librán Cuervas-Mons

Albacete, 2013
UNIVERSIDAD DE CASTILLA-LA MANCHA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS
Departamento de Ciencia y Tecnología Agroforestal y Genética

Alternatives to control microbiological cheese defects: Use of
aromatic plants

A dissertation presented in partial fulfillment of the requirements for
the degree of Doctor of Philosophy
Memoria presentada como requerimiento para optar al grado de
Doctor (Doctorado Internacional)

Celia Mª Librán Cuervas-Mons

Advisors: Dra. Mª Isabel Berruga Fernández
Dra. Ana Molina Casanova
Dr. Manuel Carmona Delgado

ALTERNATIVES TO CONTROL MICROBIOLOGICAL CONTENTS
CHEESE DEFECTS: USE OF AROMATIC PLANTS
CONTENTS
SUMMARY .................................................................................................1
CHAPTER 1: JUSTIFICATION......................................................................7
CHAPTER 2: OBJECTIVES.........................................................................11
CHAPTER 3: INTRODUCTION...................................................................15
3.1.CAUSESOFCOMMONCHEESEDEFECTS...............................................................17
3.2.MICROBIOLOGICALDEFECTSONHARDANDSEMI-HARDCHEESES...................20
3.2.1.EarlyCheeseBlowing...........................................................................................27
3.2.2.LateCheeseBlowing............................................................................................29
3.2.3.Internalmoldpresenceoncheeses.................................................................30
3.2.4.Techniquesemployedtocontrolmicrobialcheesedefects.......................33
3.3.AROMATICPLANTS...................................................................................................37
3.3.1.Extractiontechniquesofaromaticplants........................................................39
3.3.2.Propertiesanduses...............................................................................................46
3.3.2.1.Flavoringandcoloringproperties...............................................................47
3.3.2.2.Antioxidantproperties...................................................................................48
3.3.2.3.Antimicrobialpropertiesanditseffectivenesinfoodmatrixes.............49
CHAPTER 4: WORK PLAN........................................................................71
CHAPTER 5: RESULTS................................................................................79
5.1.ANTIMICROBIALACTIVITYOFAROMATICPLANTSEXTRACTS..............................80
5.1.1.Extendedsummary...............................................................................................83
5.2.EFFECTOFESSENTIALOILSINDAIRYMATRIXES...................................................117
5.2.1.Extendedsummary.............................................................................................118
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5.3.ANTIFUNGALPROPERTIESOFSAFRANAL..............................................................159
5.3.1.Extendedsummary.............................................................................................160
CHAPTER 6: DISCUSSION......................................................................179
CHAPTER 7: CONCLUSIONS.................................................................191
CHAPTER 8: LITERATURE CITED..............................................................199
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ALTERNATIVES TO CONTROL MICROBIOLOGICAL CONTENTS
CHEESE DEFECTS: USE OF AROMATIC PLANTS
TABLE INDEX
Table 1.Cheeses classificationbasedonrheological properties................16
Table 2.Maincauses of defects oncheeses andstages involved.............19
Table 3.Humanpathogenic microorganismsfoundinmilkandcheese,
source of contamination,diseases producedandways of control
microorganisms’growth.....................................................................................22
Table 4.Microorganisms assayedinantimicrobial activitytestof aromatic
plantextracts.......................................................................................................51
Table 5. Applicationofaromatic plants or itsextracts todairyproducts for
microbial growthcontrol....................................................................................68
Table 6. Antimicrobial activityresults expressedas C (μL/mL) and C
18mm 50%
(μL/mL) in case of bacteria and mold inhibition, respectively ..................... 84
FIGURE INDEX
Figure 1.Top tencountries of ewes’cheese productionin2010.................17
Figure 2.Diagramof mainsubstrates incheese fermentedbyspoilage
microorganisms andmetabolites produced..................................................24
Figure 3.Manchegocheese affectedbyearlycheese blowing................28
Figure 4.Crackedcheese affectedbylatecheese blowing......................30
Figure 5.Hardcheeseaffectedbyinternal moldpresence.........................31
Figure 6.Conidiophores of P. verrucosumvar.cyclopium............................32
Figure 7.Terpenes’structure basedinisoprene units.....................................38
Figure 8.MAPs extractionmethodologyusedandobtainedproducts......40
Figure 9. Aromatic plantdistillationmethodologies.......................................42
Figure 10.Overlappedsituationof MAPs’properties andindustries that
take advantages of them..................................................................................46
Figure 11.Commoninvitroantimicrobial tests...............................................63
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Figure 12. Antimicrobial activityof essential oils byvapour phase..............63
Figure 13.Chemical structure of commonlyfoundcompoundsin
medicinal andaromatic plants (MAPs)extracts............................................64
Figure 14.Locations andmechanisms of actionof antimicrobial
compounds inbacteria......................................................................................67
Figure 15.Doctoral Thesis workplan.................................................................72
Figure 16.Multiplate agar well dilutionmethodusedfor bacteria..............74
Figure 17.Inhibitionhalomeasurementwithadigitalcaliper......................75
Figure 18. Antifungal assaybybrothdilutionmethod....................................76
Figure 19.Myceliumweightfromthe antimicrobial assay............................76
Figure 20.Scheme of antimicrobial activitytestondevelopedoncheese
cubes.....................................................................................................................77
Figure 21. Acidificationprocess during fortifiedcheesemaking................120
Figure 22.Visual identificationof P. verrucosum growthoninoculated
cubes fromfortifiedcheeses withEOs...........................................................121
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ALTERNATIVES TO CONTROL MICROBIOLOGICAL SUMMARY
CHEESE DEFECTS: USE OF AROMATIC PLANTS

SUMMARY

Cheeses are traditionally affected by microbiological spoilage
that produces high economical losses. On one hand, bacteria such
as coliforms or butyric acid are some of the responsible of early and
late cheese blowing, respectively. These are two cheese paste
defects characterized by the internal presence of numerous and
odorous holes. On the other hand, molds have been described as
the most important problems in cheese production. They could
affect cheese appearance, flavor and texture and they could be
even involved in human health strikes due to mycotoxins
production. Control methods available are nowadays expensive,
like bactofugation or ultrafiltration. Moreover, the use of some
substances like nitrate salts or antifungal agents are extremely
controlled or not always allowed.
This Thesis aimed to provide a natural and alternative way to
control these important microbiological cheese defects by using
aromatic plants extracts’, which are well-known for their
antimicrobial properties.
To reach this objective it was necessary to assayed in vitro a
collection of 15 plants (Achillea millefolium, Anethum graveolens,
Chamaemelun nobile, Hyssopus officinalis, Lavandula angustifolia,
Lavandula hybrida, Melisa officinalis, Ocimum basilicum, Origanum
vulgare, Pimpinella anisum, Pinus sylvestris, Rosmarinus officinalis,
Salvia officinalis, Thymus serpyllum and Thymus vulgaris) in their
essential oil (EO) and aqueous extract (AE) forms against the
microorganisms: Escherichia coli, Clostridium tyrobutyricum and
Penicillium verrucosum. After this test, the three most active extracts
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