Subsections

Moulds and yeasts


Moulds

Moulds cause spoilage of food and fodder. Some strains produce mycotoxines such as ochratoxin in coffee and in cocoa which spreads out over the entire chocolate market.
They cause off flavor in food and destroy paper, wood, drugs, cosmetics etc. Moulds can cause allergies and infections.

Mouldy coffee in Trieste
In August 2006 great amount of Robusta coffee were found to be mouldy in Triest warehouse.
The beans in Trieste are thought to have been damaged by excess moisture on transport. Bags of coffee are dumped if they contain more than five mouldy beans or 10 partially mouldy beans per 500g.

Allergies
Allergies caused by moulds however are not so frequent as they seem to be. The most important sources of allergies are: To avoid mould allergy don't get in contact with cheese like Roquefort , Camembert or Brie cheese.
Keep perishable food always refrigerated to reduce mould growth.
Don't keep restover of fruits and vegetables in the kitchen. Keep it outside of the house.
Keep away from garbage [256].

Allergies

Allergies can be a serious threat to life and can end as anaphylatic shock. The allergens in foods represent a hazard for those who suffer from allergies, that is why the most important ones are cited here:

Allergens: Preservatives: A small part of humans suffer fron allergy to preservatives. Labels like "Free of preservatives" must be true as some persons suffer heavy allergic responds to some preservatives. To avoid recourses due to cross over " No preservatives added" is being now labeled. This, however, does not solve the problem of allergic reactions.


Culture media for moulds and yeasts

Moulds and yeasts spoil foods. It is therefore important to control raw materials and finished. A useful medium is the Yeast Chloranphenicol dextrose Agar. Moulds grow as large colonies and are easily identified. Yeasts grow as small colonies, Both types of microorganism can grow with beautiful colors.

Moulds can be phytopathogen and can cause serious damage to agriculture.
Moulds have also a good side. They produce antibiotica like Penicillin, Cephalosporin and Griseofulvin and many substances in industrial scale such as citric acid, succinic acid, glucuronic acid, and malic acid. Moulds can also be used in the production of polymer such as Pullulan. They are used to produce beta-carotene, enzymes such as amylase glucoamylase, Protease, Lipase, pectinase, cellulase, lactase, catalase some types of cheese, sausages, fermentation of certain food such as soya, rice and corn. Examples of pathogen moulds:
Gliocladium species Destroys paper, may be present in fuel.
   
Isaria species Pathogen to insects.
   
Paecilomyces species grows very quickly, infectious.
  Produces mycotoxines such as patulin and Byssochlamin acid
  in fruit and juices.The ascospores resist
  Temperatures up to 85$^{o}$C during 30 minutes
  96$^{o}$, the plant must be dismantled
  to get rid of Paecilomyces.
   
Scopulariopsis brevicaulis grows quickly,sometimes pathogenic.
  pH optimum 9 - 10,grows in tilsit cheese and camembert cheese
  and meat and derivates
   
Trichoderma species Produces trichothecenes and T-2-toxin
  grows at pH 2.5 up to 9.5 and can
  grow on sour food, is found an corn, rice and wheat.
   
Verticillium species pathogenic for plants.


Selective culture of Candida albicans

The selective culture of Candida albicans uses Merck Fluoroplate Candida -Agar (1.11011) . It is a modified SABOURAUD with the addition of fluorogene substrate (MUGal) which can be hydrolysed by the enzyme Galactosaminidase resulting a fluorescent compound. 99% of Candida albicans bear this enzyme. Incubate at 37$^{o}$C for 2-3 days. Read the plates under UV light. All fluorescent colonies are Candida albicans.

Candida albicans ID:

Another selective and chromogenic medium for identification of Candida albicans is the albicans ID medium bio Mérieux Nr.43 121 citealbicans

Ingredient Amount for 1.000 ml
   
bio-Thione 2,00 g
Yeast extract 6,00 g
Monopotassium phosphate 0,5 g
Dipotassium phosphate 0,5 g
Chromogen substrate (hexosamine) 0,05 g
ADA butter 0,6 g
Gentamicin sulfate 0,10 g
Chloranphenicol 0,05
Agar 14,00 g

pH 6.6

Albicans ID is a medium to isolate yeasts and immediately identify Candida. Colonies of Candida albicans grow as blue colonies on Albicans ID medium due to hydrolysis of hexosaminidase chromogen substrate. The two antibiotic which are present in albicans ID inhibit the bacterial flora. The buffer system of the medium facilitates the growth of yeasts and optimizes the enzyme reaction.
Albicans ID medium is used to detect yeasts by direct inoculation of pathological specimens (buccal, vaginal, rectal swabs,feces, scales, pus, urine. It should also be suitable to control food),

Reading of the culture is made after incubation at 30 to 37$^{o}$C for 24 to 48 hours. The colonies have a round, smooth,slightly domed shape, with a clearly defined border, and blue coloration. This coloration does not diffuse into the medium and turns from pale to dark blue depending on the incubation time.The size of the colonies is 0.5 to 1.5 mm.
Other yeast species have a creamy-white color and the size is 0.25 to 2 mm. A complete biochemical identification should be carried out if required.

Albicans ID medium must always be stored and incubated in the dark.

Some strains of yeasts such as Saccharomyces cerevisiae and Cryptococcus neoformansgive small colonies and require an incubation of more than 48 hours.

A small percentage of Candida tropicalis strains give blue colonies within 48 hours.

Certain strains of Trichosporon cutaneum can give cotton-like bluey-green colonies, which can be easily differentiated from Candida albicans colonies.

More attention should be payed to Candida albicans on bacteriological control of food as this yeast can cause diseases of the intestinal tract.
The Fluoroplate Candida-Agar contains chloranphenicol and gentamycine to inhibit accompanying bacteria.
The isolation can be made directly from faecis or vaginal samples.
Moulds attack human when there is a lesion such as burns, frost, injuries, immuno insufficiency, serious diseases such as tumor, diabetes, leucosis, transplantations, use of immunosupresives, therapy with antibiotic.

Growing conditions

Molds can grow at a wide rage of temperature:

Growth of mycelium Temperature
optimum growth of mycelium 25-30$^{o}$C
maximum growth of mycelium 30-40$^{0}$C
As example Penicillium optimum 20-25$^{o}$C
As example Aspergillus optimum 25-35$^{o}$C


Minimum growth of mycelium 0$^{0}$C
optimum growth of mycelium 30-40$^{0}$C
maximum growth of mycelium 50$^{0}$C
As example Mucorales optimum 30-41$^{0}$C


Some moulds may grow under 0$^{0}$C ,
such as Chladosporium growing at -6$^{0}$C.

Minimum growth of mycelium 25$^{0}$C
optimum growth of mycelium 35-45$^{0}$C
maximum growth of mycelium 55-60$^{0}$C
As example Rhizomucor pusillus  


Temperatures for best production of toxins differ often from growth optimum
Spores and sclerotias can survive high temperatures.
Only molds which can grow at 37$^{0}$C can cause a systemic mycose.

pH-optimum 4.5 - 6.5
pH-maximum in the majority 8.0
  Exceptions are 9.8 - 10.5 such as:
  Aspergillus niger,
  Penicillium italicum,
  Aspergillus flavus.
pH-minimum for growth of moulds 2.0 and below


Moulds are aerobic They grow however also as microaerobic.
Many Mucorales can produce fermentation.  
Increasing CO$_{2}$ reduces  
growth of many moulds.  
At the same time reducing oxygen  
stops completely growth of molds on fruits.  

Culture and microscopy of moulds

The morphology of colonies vary with the culture media employed, age of the culture, the species and the temperature of incubation.
Preparations for microscopy should be made with 50% alcohol because moulds are not water soluble. Don't use very old colonies because only spores are present.


Black moulds

Black moulds have their color due to melanin in their mycelium.
They are UV-resistant and very frequent in the environment. They destroy materials and spoil food. They are potential allergens.

Determination of yeasts and moulds

Optimal media for the determination of yeasts and moulds
Wort agar and Malt extract agar
Selective media

DRBC (Dichloran-Rosebengal-Chloramphenicol-agar): . This culture medium is suitable for fresh food with high water activity. It contains 25 ml/l Rosebengal and 2 mg/l Dichloran. Culture on surface and incubation at 25$^{o}$ for 5 days.

DG 18 (Dichloran 18% glycerol agar): This medium is suitable for xerophile moulds from cereals, nuts, flour and spices.

OGY (Oxytetracyclin glucose yeast extract agar: This medium is suitable for determination of yeasts and moulds simultaneously.

Fresh moulds DRBC on surface
food yeasts TGY pourplate
    MEA, OGY  
  yeasts and DRBC  
  moulds    
       
Nuts,cereals yeasts and DRBC on surface
  moulds    
       
Fruit juice yeasts TGY, MEA, OGY pourplate
       
Concentrated xerophile MY50G pourplate
fruits yeasts    
       
Dried food yeasts and DG18 on surface
  moulds    
       
dried fruits yeasts MY50G on surface
and chocolate molds    
  xerophile    
       
All samples aflatoxins AFPA  
on surface or     pourplate
       
All samples Ochratoxin DRYS on surface or
      pourplate


DRBC= Dichloran-Rosebengal-Chloranphenicol-Agar
TGY= Tryptone glucose yeast extract agar
MEA= Maltextract-agar
OGY= Oxytetracyclin glucose yeast extract agar
MY50G = Malt extract agar, 50% glucose
DG18= Dichloran, 18% Glycerol
AFPA= Aspergillus flavus parasiticus agar
DRYS= Dichloran rosebengal yeast extract saccharose agar


Contamination of cereals with Fusarium graminearum and Fusarium culmorum

According to a definition of the food regulation of the EU cereal grains are to be classifies as unusable when :
Mycotoxines are present.
The value of the grains is reduced because of bacterial activity.
There are changes of smell and color
Bacterial count is high
Tolerated by EU regulation are 0,5% of grains with black Fusarium contamination.


Trichotecene


Zearalenon

Zearalenon is a mycotoxin which can be present in animal feed and can cause be found in muscles and organs of animals with destination as human food.


Citrinin


Patulin

Patulin is frequently produced by Penicillium expansum on fruit juices when sterilization is using spoiled fruits.

Very important for the production of juices is to select rotten and mouldy fruits on the transportation belt, use fresh and not contaminated water, a brush station for some kind of fruits. extreme care should done to keep all machines and the surroundings always clean to avoid the growths of bacteria and moulds. Be always aware that the bacteria and moulds can be killed by sterilization, the poisons such as patulin however are not inactivated by heat.Quality and safety of food depend on careful handling through the whole production process.

Pasteurization of fruit juicesshould be done at 60 to 90$^{o}$C, orange juice at 85 to 90$^{o}$C for several minutes. Enzymes such pectin esterase are also inactivated during this procedure. Yeasts which can spoil wrong treated juices are Candida, Cryptococcus, Hanseniaspora, Rhodotorula and Saccharomyces cerevisiae Common moulds on fruit juices are Geotrichum, Mucor, Penicillium and Phialophora.

Fruits as raw ware for juice industry should have not more than 2 X 10$^{6}$ yeasts and not more than 2X 10$^{5}$ moulds/g otherwise alcohol and toxic products are formed.[712]. Talaromyces trachyspermus, Talaromyces flavus and Neosartorya fischeri are moulds which may develop heat resistant organisms an may spoil juices during storage.

In storage tanks dripping water from condensation may dissolve the juice at that point making it possible for moulds and yeasts to grow.
Green colonies may be the result of growth of Penicillium expansum, black colonies of Aspergillus niger. One percent of N$_{2}$ of the atmosphere of storage tanks may be useful to avoid growing of moulds.
Bottles and other packaging materials should have less than 1 mikroorganism/cm$^{2}$.
Layer yeasts such as Candida boidinii,Candida intermedia, Candida parapsilosis and Debaryomyces hansenii produce a slimy yeast skin.


Measures to reduce the risk of contamination of juices with yeasts and moulds

Use good raw material, not spoiled, with low bacterial count.
Look out for proper cleaning and disinfection of the equipment.
Avoid underheat of the juice.
Use 2% of N$_{2}$or CO$_{2}$ in the atmosphere of storage tanks.
Cool storage tanks down to 2$^{o}$C.
Avoid dripping of condensation water in storage tanks.
Use bacterial filter on air inlet of tanks.


Ryegrass staggers:

[1027][1028] Feed grass such as lolium (Lollium perenne which grows in North America and Australia also called "English ryegrass" and festuca (such as Festuca ovina) may have a symbiotic community with a mould which rises the vitality of the gras, reducing drought damage and resistance to various pests.
The mould may produce under certain weather conditions alkaloids such as lolitrem B which may have an adverse effect on the cattle.This toxicosis is called "ryegrass staggers" and is common in North America and in New Zealand and in some cases also in Europe.


Moulds and bacteria found in spices

Spices grow in tropics and subtropics and are therefore submitted to ideal temperatures and humidity conditions for the growth of bacteria and moulds.

Lactic acid bacteria against Fusarium mycotoxins[1029]
According to Biotechnology Ireland the study of specific lactic acid bacteria (LAB) may help to reduce Fusarium mycotoxins and ochratoxinin in grain production. New legislative requirements for the reduction of mycotoxin content in cereal-based products demand for alternative methods to prevent mould growth.

Scientists at the University College Cork are screening food grade la Yeasts [1030]
For practical purposes, yeasts may be defined as unicellular fungi in which assexual reproduction occurs mainly by budding. Budding was defined by von Arx 1979, as a type of conidiation and the buds are blastoconidia. Yeasts are characterized, classified and identified traditionally by morphological, physiological, and biochemical criteria

Yeasts area phylogenetically diverse group of fungi. Their sexual states (teleomorphs) can be classified among two major fungal classes, the Ascomycetes and the Basidiomycetes.

A classification should also consider genetic similarities and differences. The application of molecular biology has already made a large impact on the systematics of yeasts.

Yeasts are generally unicellular, some may, however, develop hyphae or pseudohyphae. True hyphaelack constriction at the cross walls, pseudohyphae cells are formed by budding and elongation. [1031]

Yeast-like organis: Buds arising from true hiphae. Hyphae may separate into arthoconidia. Both filamentous forms are collectively called yeast-like organism.

Growth temperature: Most yeasts do not groiw under 0 $^{o}C$, psychrophilic can grow at temperature which go down to -7$^{o}$C. Freezing and subsequent thawing can cause loss of viability. Lethality is reduced when cells are rapidly frozen and rapidly thawed. The degradation of membrane phospholipids and cell water permeability may cause the death to yeast cells subjected to freezing and thawing. [1032] [1033]

Candida albicans: Yeasts are mainly known to have impact on food spoilage, formation of haze, sediment and off-flavour in carbonated drinks and juices, as well as bulging cans and exploding bottles. There are, however, some yeasts which are pathogen to humans such as Candida albicans.
Candida albicans can be diagnosed by the formation of germ tubes and clamydospores. Candida albicans can be found in foods such as soft drinks, must, wine and others. Candida tropicalis is also present on food and should be diferentiated from Candida albicans.

Differentiation of Candida albicans and Candida tropicalis
Yeast sucrose maltose
Candida albicans - +
Candida tropicalis + +



Molecular biology and Classification
Studies on DNA base composition, nuclear DNA homology, and sequences of ribosomal RNA are used to elucidate the degree of relatedness and evolutionary relationship of yeasts. These methods, however cannot be easily applied in routine identification procedures, therefore, the classification of yeasts is still primarily based on characteristics of sexual reproduction.

The species Saccharomyces cerevisiae, for instance, described by Hansen in 1888, suffered important changes and in 1985 , 1987 and 1989 Vaughan-Martini and Kurtzman separated the genus Saccharomyces in four genra: S. cerevisiae, S bayanus and S. pastorianus which are used in industrial fermenting. A fourth genus S. paradoxus has no relation to alcoholic fermentation. [1034] [1035] [1036] [1037]

Saccharomyces cerevisiae: Is the leavening of bread and the fermenter of alkoholic beverages. Saccharomyces cerevisiae has one or more genes coding for alfa-glucosidase and maltose permease.

Chalky bread
Yeasts and yeast-like organisms may develop white spots in the crumb, which is called chalky bread. According to Spicher (1986), envolved in chalky bread are Endomycopsis fibuliger, Pichia burtonii, Zygosaccharomyces bailii, Saccharomyces cerevisiae, Torulaspora delbrueckii, Pichia membranaefaciens and Candida parapsilosis. [1038] [1039]

Spoilage of soft drinks Spoilage of carbonated soft drinks are most frequently being caused by Saccharomyces cerevisiae and Saccharomyces pastorianus. Contamination most often results from incorrect sanitation of processing line which includes, holding tanks, proportioning pumps and bottle washers. Gas production may be very heavy leading to the explosion of the bottle. [1040] [1041]

Wine, Beer and distilled spirits
Crushed grapes which stand on the start of the production line of wine contain species like Hanseniaspora uvarum, Candida stellata, Issathenkia orientalis, Metschnikowia pulcherrima and Pichia anomala. [1042] [1043]

According to Fleet a definite succession of yeasts takes place during the fermentation under the influence of growing alcohol. The non-Saccharomyces species die off with an increase of ethanol content. These yeasts are more tolerant to alcohol at lower fermentation temperatures [1044] The population of yeasts in the fermentation phase depends on the variations in the ecosystem of different vineyards. Hanseniaspora uvarum in Middle-Europe, Japan and California whereas Hanseniaspora osmophila in warmer regions like Italy, Israel or Southern US. Dominate the early fermentation. [1045]

According to Martini Kloeckera apiculata initiates fermentation and is followed by Saccharomyces cerevisiae. [1046] As ethanol concentration rises between 2 to 6% wild yeasts die and Saccharomyces cerevisiae dominates till the fermentation is complete. Inoculating strains of Saccharomyces cerevisiae at the beginning of the fermentation does not change this sequence. [1047]
After the fermentation any further yeast activity harms the quality of the product. Yeasts are therefore removed by racking, filtration and other cellar. Dekkera species, Saccharomycodes ludwiglii may impair flavour of bulk wine. Candida vini, Candida zeylanoides, Candida rugosa, Issatchenkia orientalis, and Pichia membranaefaciens are responsible for spoilage of wine in tanks and in wooden barrels. They are film-forming species. [1048] [1049]

Spoilage of bottled wines: Bottled wines, according to Minarik, are often spoiled by Zygosaccharomyces bailii (considered as the main cause of spoilage), Saccharomyces cerevisiae, Candida rugosa, Pichia membranaefaciens and Candida vini. Activity of these yeasts cause cloudy appearance, sediment and poor flavour. [1050]

Beer Lager beer is fermented by Saccharomyces pastorianus. This strain is a bottom fermenter and produces alfa-galactosidase, hydrolysing melibiose and raffinose. The fructose part of the raffinose molecule is fermented. The yeasts flocculate and settle on the bottom of the fermenter. Fermentation takes place only up to 34$^{0}$C. Ale beer is being produced with Saccharomyces cerevisiae which is unable to ferment melibiose and only the fructose part of the raffinose molecule is fermented. It tends less to flocculate than S. pastorianus. Fermentation of sugars takes place up to 38$^{0}$C. [1051]

Silage:Silage is an important part of animal feed in northern regions. It is a product of a lactic acid fermentation. Yeasts may cause alcoholic fermentation. According to Engel, the most frequent yeasts which may spoil silage are Pichia fermentans, Issatchenkia orientalis, Saccharomyces cerevisiae and Geotrichium candidum [1052] [1053]. These yeasts assimilate lactic and acetic acid, resulting in silage spoilage if exposed to oxygen of air.

Fermentation of cocoa beans:Yeasts, together with lactic acid bacteria present the traditional cocoa fermentation process for removing the mucilaginous pulp around the seed. Sanches found the following species of yeasts to be involved in this process: Hanseniaspora uvarum, Saccharomyces cerevisiae, Pichia membranaefaciens, Pichia fermentans and Issatchenkia orientalis.[1054]

Yeasts identification methods [1055]

Various systems of identification of food-borne yeasts have been developed. Deak compared three yeast identification methods:

The Simplified Identification Method (SIM) (identifying 91% of the samples correctly), the commercial kits, the Analytab API 20X (86% correct identification) and the BioMerieux Vitek Yeast ID 32C strips(76% correct identifications).

Discrepant test reactions and errors in the database had caused the false identifications. Whereas, the accuracy of individual test were high with discrepant results of 1,6%, 2,5% and 1,7% respectively. Deak notes, therefore, that with a supplement of a few tests, the described commercial kits can be easily applied to the SIM database.
Other commercial systems for the identification of food-borne yeasts are Minitek, AutoMicrobic, ATB 32 ID YeastIdent.

Based on foregoing experiences Deak suggests a revised SIM version which includes 99 of the most common food-borne yeasts, and 30 tests are applied. In the SIM key, great reliance is given to sugar and nitrogen assimilation tests. As the keys uses tests in which yeasts give 85% to 100% unequivocal responses, a certain probability exists that the results do not fit the identification scheme. The author stresses therefore that species identification should never be based only on those features included in the keys.

DNA identification
A technique revealing restriction fragment length polymorphism (RFLP) have been used in taxonomic evaluation of yeasts. It is based on the activity of restriction enzymes which generate numerous fragments of variable length, resulting in characteristic banding patterns. Degré found that DNA fingerprinting, using RFLP found it to be the most reliable method to identify brewers yeast strains. [1056]

Casey, however, using RFLP, could not differentiate ale from bakers yeasts and concluded that RLFP pattern not to be suitable as an universal method to identify different strains within the same yeast species. [1057].

Electrophoretic Kariotyping
Electrophoretic Kariotyping determines the chromosomal number and size which can be achieved by electrophoretic separation of whole chromosomes in agarose gel.

Török used whole chromosomes from yeasts as templates for probe preparation to distinguish between closely related yeast species. [1058] [1059]

DNA Probes and PCR Amplifications
The PCR technique and specific probes are promising as reliable and rapid molecular methods for the identification of yeasts.

Protein Elektrophoresis
Soluble protein electrophoresis using polyacrylamide gel, sometimes with sodium dodecyl sulfate was used by Bruneau and Giunet to identify medical important yeasts. [1060]

Degré, however, found contradictory results using this technique depending on growth conditions. Degré concluded that the reproducibility of protein and fatty acids patterns requires rigidly standardized methods. [1056]

Fatty acid Analysis
It uses gaschromatography of cellular volatile fatty acids. A commercial identification system of fatty acids from yeasts has been developed by MIDI. [1061]

Augustyn, however, using the fatty acid analysis technique, could not differentiate between Saccharomyces cerevisia, Saccharomyces bayanus and Saccharomyces pastorianus, important for the beer and wine brewery. [1062]

Rozes found that the method has potentials for distinguishing fermenting wine yeasts from spoilage yeasts. [1063]

Humanised yeast cells [1064] [1065]
In 2000 Tillman U. Gerngross co-founded GlycoFi, Inc., base in Lebanon, It is a company pioneering the "humanization" of yeast and fungal protein expression systems.
Professor Gerngross research examines both the macroscopic and the microscopic scale of biotechnology.

Protein-based therapies have to be manufactured by living cells, which are genetically engineered to produce a given protein of interest often requiring the attachment of sugar structures (glycosylation). This could only be performed in mammalian cells.

Human glycosylation can now take place within a yeast cells of Pichia pastoris, eliminating the need for mammalian cells. The new technique reduces the risk of contamination by pathogens and infectious agents.

According to Stephen Hamilton from GlycoFi, humanizing the glycosylation in yeast required the silencing of four yeast genes and the introduction of over 14 heterologous genes.

With this engineered yeast some glycoproteins were produced such as erythropoietin, used to treat anemia, and other glycoproteins such as antibodies with improved anti-cancer properties.

The study details the genetic engineering of the yeast to secrete human glycoproteins with fully complex, terminally sialyated N-glycans.

The authors conclude that the ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.

According to Y. Ma and colleagues, Pichia pastoris is already being used to elaborate the human adenovirus type 5 (Ad5) early-region 1A (E1A) proteins which have strong tumor-suppressive activities in human tumor cells. The authors stress that the E1A protein overcame the limitations of gene therapy and may be a useful therapeutic agent for some malignant tumors. [1066]

Identification of yeasts

[1122] Identification can be performed either phenotypically usind fermentation reactions of sugars or growth on carbon and nitrogen sources or other compounds. These characteristics can vary, depending the physiological state of the cell. Molecular biology techniques are independent of the state of the cell as they analyse the genome of the cell.

The nucleotide sequences of the domains D1 and D2 located at the 5' end of gene 26S (Kurtzman and Robnett, 1998) and PCR amplification of ribosomal DNA regions and restriction of the gene 5.8S rRNA gene and the adjacent intergenic regions ITS1 and ITS2 are the molecular methods commonly used for the identification of yeasts (Fernandez-Espinar et al.,2006). These techniques are more reproducible and faster that the conventional methods based on physiological and morphological characteristics. [1124]

Yeasts patogenic for human [1123] The principal yeasts pathogenic for humans are Candida albicans and Cryptococcus neoformans which cause a range of mucocutaneous, cutaneous, respiratory, central nervous, systemic and organ infections.. Usually, healthy, immunocompetent individuals are not at risk of such infections. Generally, individuals with weakened health and immune function are at greatest risk, and include cancer and AIDS patients, hospitalised patients and patients who are administered immunosuppressive drugs, broad-spectrum bacterial antibiotics and radio- and chemotherapies.

This includes species that are frequently found in food such as Candida krusei/orientalis, P. anomala, Kluy. marxianus, S. cerevisiae and various Rhodotorula.

EFSA Qualified Presumption of Safety (QPS) of Yeasts [1123]

Candida The genus Candida comprises 163 species, of which around 60 species are present in food. Only a small number of Candida are used in food processing, as biocontrol agents such as C. glabrata is used to control filamentous fungi in plants.

The list of species that are commonly used in the food industry include: C. zelanoydes, which contributes to the flavour and texture during the maturation of cheese and in the production of fermented milks (kefir and koumiss), C. milleri for flavour and rheology in sourdough breads, C. tropicalis, C. parapsilopsis produces skin lesions and C. pelliculosa, which occur in the wet fermentation of coffee, C. etchellsii and C. versatilis, which contribute to the flavour of soy sauce, C. rugosa, which is involved in cocoa fermentations, C. utilis (=P. haidinii) and C. maltosa, which are used for biomass production from carbohydrate and hydrocarbon substrates respectively, C. oleophila and C. sake, which are commercialised for use as fungal biocontrol agents.

Pathogenic Candida spp.

The principal human pathogenic yeasts are species of Candida, such as C. albicans, C. glabrata , both as most frequent pathogenic yeasts. C. guilliermondii, C. krusei, C. lusitaniae, C. parasilopsis, C. tropicalis produces deep seated micoses, C. viswanathiin and new emerging pathogen is C. dubliniensis.

The Candida genus is not suitable for QPS status, as more species are today considered as emergent pathogens.


Debaryomyces

The genus Debaryomyces comprises 15 species. Many representatives can be isolated from natural habitats such as air, soil, pollen, tree exudates, plants, fruits, insects, and faeces and gut of vertebrates.

Nine of these Debaryomyces species: D. carsonii, D. etchellsii, D. hansenii, D. maramus, D. melissophilus, D. polymorphus, D. pseudopolymorphus, D. robertsiae and D. vanrijiae, have been found in a variety of processed foods; such as fruit juices and soft drinks, wine, beer, sugary products, bakery products, dairy products and meat or processed meats. The presence of Debaryomyces species in foods usually has no detrimental effects and in some cases is beneficial to the food.

Some Debaryomyces species are important in the ripening of fermented food products such as cheese and meat products. Where D. hansenii is used in the ripening of cheeses they metabolise lactic acid, raising the pH to allow the growth of proteolytic bacteria, and the yeast exhibits lipolytic activity that contributes to the development of cheese aromas. Proteolytic and lipolytic activities of D. hansenii have been described in the curing of ham and ripening of sausages and their presence in salami influences the red coloration and improves the quality of the product.

Nevertheless, excessive growth of Debaryomyces species may cause undesirable sensory changes due to the formation of off-odours and off-flavours. These species have also been found as frequent contaminants of spoiled yoghurts, ice creams, fish, shellfish, etc.

Very often, fungi have got two forms : the sexual form (teleomorph) is considered as perfect while the asexual form (anamorph) is considered as imperfect form. The Fungi imperfecti are only known by their asexual form (Conidia). [1125]

The main species of Debaryomyces used in food processing is D. hansenii, the anamorph form of which is Candida famata. C. famata has been repeatedly associated with catheter-related bloodstream infections, and occasionally with infections of the central nervous system. The reservoir of C. famata is not known but there is a possibility that nosocomial infections can occur via air contamination (Wagner et al., 2005).

No studies on antifungal susceptibility of Debaryomyces are available. It is proposed to grant D. hansenii QPS status.


Hanseniaspora

The species are most frequently isolated from soil, fruits and plant exudates, grapes and processed fruit. Hanseniaspora uvarum is important in the first phase of grape fermentation and play a role in the production of certain flavours beneficial for the quality of wine and cider. Little is known regarding the other species.H. uvarum is proposed for QPS status.


Kluyveromyces

There are six species present in this genus.The most important are K. lactis and K. marxianus (anamorph C. kefyr) for their capacity to ferment lactose. This microorganism can be isolated from milk products and is used as a starter to set up the medium for cheese and kefir production. Kluyveromyces marxianus and K. lactis are associated with smear-ripened cheeses and contribute to the aromas that cheeses develop. These species are considered to be generally regarded as safe organisms and have been approved as a food additive.

Kluyveromyces is used in animal feeds in Europe as a probiotic and is apparently safe (reviewed in Anadon et al., 2006).

Candida kefyr, the anamorph of K. marxianus, has occasionally been involved in opportunistic infections in immunocompromised persons. However, considering the history of apparent safe use and the rarity of infections in humans, there are no safety concerns. It is proposed to grant K. lactis and K. marxianus QPS status.


Pichia

Yeasts of the genus Pichia are widely distributed; they can be found in natural habitats, such as soil, freshwater, tree exudates, insects, plants and fruits, and also as contaminants in a variety of foods and beverages. Some Pichia species contribute desired effects in the early stages of wine fermentation, several types of brines, and different types of cheeses; while others have been described as human pathogens (Bakir et al., 2004; Otag et al., 2005).

Pichia currently contains 91 species with 30 being related to food production and processing, the majority of them are spoilage organisms. The genus contains the species previously encompassed in the genus Hansenula, which is reported to be one of the safest microorganisms; it is used by the WHO for the development of vaccines and as a producer organism such as phytases.

The main species are P. anomala (previously Hansenula anomala) and P. angusta (previously Hansenula polymorpha). P. anomala is also used for the fermentation of bakery products, while P. roqueforti is used as a post-harvest biocontrol agent for wheat and barley, or for food application in olive fermentations.

Some species of Pichia are used for feed (source of proteins) and production of glucan for feed. It is proposed that P. angusta and P. anomala have QPS status.


Saccharomyces

These species are strongly fermentative, and are commonly isolated from soil, fruits, foods and beverages. S. cerevisiae, S. pastorianus and S. bayanus are widely used for making bread and in the production of beer, wine, distilled beverages and fuel alcohol. S. cerevisiae occurs on fruit, in processed fruits, dairy products and plays a role in the fermentation of kefir, coffee, cocoa, and the production of traditional fermented products. S. cerevisiae and S. bayanus cause spoilage of soft drinks.

In one review, cases of Saccharomyces invasive infection were presented (Enache-Angoulvant and Hennequin, 2005). Predisposing factors were similar to those of invasive candidosis, with intravascular and antibiotic therapy being the most frequent. Blood was the most frequent site of isolation. S. cerevisiae (subtype S. boulardii) accounted for 51.3% of fungaemias and was exclusively isolated from blood. Special caution should be taken regarding the use of S. cerevisiae (subtype S. boulardii) preparations (Fleet and Roostita, 2006). There are number of recent reports and reviews regarding the safety of S. cerevisiae (subtype S. boulardii). The authors concluded that probiotics should be used cautiously in certain high-risk populations.

A review of the current literature reinforces the view that fungaemia and sepsis are rare complications of the administration of S. cerevisiae (subtype S. boulardii) in immunocompromised patients but confirms that the most important risk factor for S. cerevisiae fungaemia is the use of probiotics (Herbrecht and Nivoix, 2005; Munoz et al., 2005).

This raises the question of the risk-benefit ratio of these agents in critically ill or immunocompromised patients who are likely to develop an infection after exposure to high amounts of a microorganism with a low virulence. The authors concluded that S. cerevisiae (subtype S. boulardii) should certainly be contraindicated for patients of fragile health, as well as for patients with a central venous catheter in place. It is recommended that a specific protocol concerning the use of probiotics needs to be formulated.

It is possible to propose some species of the genus for QPS status with the following qualification: "provided the proposed species does not grow at 42$^{o}$C and is not filamentous" S. bayanus, S. cerevisiae and S. pastorianus (syn of S. carlsbergensis) are proposed for QPS status with the above qualification.


Schizosaccharomyces

Three species are included in this genus, Sch. japonicus, Sch. octosporus and Sch. Pombe, living on fruits and fruit juices, wines, tequila fermentation and high sugar concentration. They are strong fermenters of sugars and have been used for the production of ethanol.

The species Sch. pombe is used as a phytase producer for animal feed; minimum safety precautions should be taken for the handling and storage. No infection issues have been reported. Sch. pombe is proposed for QPS status.


Xanthophyllomyces

Phaffia rhodozyma ferments D-glucose and occurs in slime fluxes of deciduous trees. The anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous forms are known.

The yeast is used to synthesize the carotenoid astaxanthin (3,3'-dihydroxy-β, β-carotene-4,4'-dione), a dietary source for aquaculture and poultry industries, including salmonids, lobsters and the egg yolks of chickens and quail. Xanthophyllomyces dendrorhous is proposed for QPS status.

Fungal DNA and galactomannan and glucan antigens as screening of high-risc patients

[1126]
Accordingt to Jones and McLintock (2003) reduction of the mortality of invasive fungal infection could depend on the development of rapid, sensitive diagnostic methods such as serological and molecular techniques, assessing the utility of these methods and consider their role in management strategies.

The authors propose the detection of fungal DNA and antigens such as galactomannan and glucan which have been prospectively evaluated in the clinical setting in early diagnosis of invasive fungal infection of high-risk patients.

The sensitivity and specificity of the assays depends on patient selection, clinical application of the test, and release and circulation of galactomannan and fungal DNA.

The authors conclude that it is essential these tests to be incorporated into management strategies and call for further clinical trials.

Invasive aspergillosis and galactomannan test [1127]

According to Gonzalo Bearman the invasive aspergillosis is acquired by inhalation of airborne conidia by susceptible host. Its incidence is 5 out of 100,000 people and can develop:
Pulmonary aspergillosis(most common), CNS aspergillosis, sinonasalaspergillosis, osteomyelitis endophthalmitis, endocarditis, renal abscesses, cutaneous.

Definitive diagnosis requires the demonstration of tissue invasion, and positive culture from biopsy specimen. Invasive tissue biopsy is often impossible due the debilitated state of the patient. Less or non-invasive tests that may suggest the diagnosis.are isolation of Aspergillus from sputum and testing for galactomannans. Serologic Aspergillusprecipitin assays are rarely elevated in IA.

Testing for Galactomannans

[1127]
Galactomannan is a component of the fungal cell wall and an exoantigen of Aspergillus. PlateliaTM AspergillusEIA[1128] was approved by the FDA in 2003 and detects galactomannan in serum. In the dataset evaluated by FDA, the overall sensitivity and specificity of the method were 80.7% and 89.2%, respectively. However, Bearmann found that significant test variability has been reported post marketing which show a sensitivity 29-100% and specificity $>$85% . The test performance is likely affected by: in vivo characteristics of galactomannan secretion of Aspergillus the patient population, and other factors.

Antigenic cross reactivity with other fungi such as Penicillium chrysogenum, Penicillium digitatum, and Paecilomyces variotii may result in false positive testing.

Conclusion

Bearman concludes that the use and interpretation of PlateliaTM AspergillusEIA may be of some, yet limited, value in the diagnosis of IA in high risk patients.

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