Food Microflora – Molds

What are Molds?

• Foods with mold growth, which can sometimes be colored and fuzzy or cottony appearance, are common to all. Foods moldy or “mildewed,” are usually unfit for consumption.
• Although molds can cause spoilage in many foods, some molds can be used to make certain food or ingredient.
• Some types of cheese can be moldripened in this way, such as blue, Roquefort, Brie, Gammelost, and Camembert. Also, molds can be used to make Oriental foods like soy sauce, miso and arei.
• The molds can be grown for food or feed and used to make products such as amylase in breadmaking or citric acids in soft drinks.

• The term mold is used to describe multicellular, filamentous fungi that can be easily identified by their fuzzy or cottony appearance.
• The majority of the growth appears white, but it can also be darkened or smoky.
• The color of colored spores is a sign of mature mold and can be used to give color to some or all of the growth.
• The thallophytes lack true roots, stems and leaves, so the thallus is a characteristic of them.
• Molds are filamentous fungi. They grow quickly and cover many inches in a matter of days.
• Mycelium is the sum of all or a large part of the mass.
• Mycelium is made up of branches and filaments, also known as hyphae.
• The most important foods are those that multiply via conidia, zygospores or ascospores.
• Some genera’ ascospores are known for their extreme heat resistance.
• One group is pycnidia (or acervuli) which are small, flask-shaped fruiting bodies lined by conidiophores.
• Arthrospores are formed when hyphae fragment in certain groups.
• In1980s there were no major changes in the foodborne fungi system. Most notable were the discoveries of sexual or perfect states in some well-known genera.
• Mycologists believe that the ascomycete state, which is also known as the teleomorph, is the most important reproductive state for a fungus.
• The species name for a Teleomorph is more important than that given to an Anamorph, the imperfect or conidial.
• Holomorph means that both states exist, but the teleomorph is used.

Morphological Characteristics of Molds

In their classification and identification, they are based on their morphology (i.e. their form and structure as determined by their microscopic and macroscopic appearance).

1. Hyphae and Mycelium of Molds

• Mold thallus is composed of a mass of branching, interconnected filaments, called hyphae (single hypha), and the entire mass is known as mycelium.
• The hyphae can be submerged or grow within the food or aerial or grow into the air above the food.
• Hyphae can also be classified as vegetative or growing and are therefore involved in the nutrition of the mold, or fertile in the production of reproductive parts.
• The fertile, hyphae in most molds are usually aerial. However, they can be submerged in some molds.
• Some molds have hyphae that are smooth and full, while others have hyphae that are ragged and thin.
• Some molds can produce sclerotia, which is a single-sclerotium, which are densely packed masses of modified Hyphae that are often thick-walled within the mycelium.
• These sclerotia have a higher resistance to heat and other adverse conditions than the rest of mycelium. This may make them important in certain processed foods.
• The microscopic examinations of mold hyphae reveal characteristics that can be used to identify genera.
• There are two types of molds:
  • septate, i.e., with cross walls dividing the hypha into cells; and   
  • noncoenocytic, septate with the hyphae apparently consisting of cylinders without cross walls.
• Nonseptate hyphae are multicellular and have nuclei distributed along their length. Most molds have clear hyphae, while some may be darkened or smoky. Although hyphae can appear transparent and uncolored on microscopic inspection, they can be colored when large numbers of them are viewed macroscopically.
• The length of septate hyphae increases by the division of tip cells (apical) or of cells within hyphae (intercalary), depending on the type of mold.
• The increase of filament length is associated with the division of nuclei distributed through nonseptate Hyphae.
• Mycelial structures are part of the identification of molds. The rhizoids (or “holdfasts”) of Rhizopus, Absidia, and the foot cell In Aspergillus are just a few examples. Also, Geotrichum’s dichotomous or Y-shaped branching is another example.

2. Reproductive Parts or Structures  of Molds

• A transplanted bit of mycelium can help molds grow.
• Asexual spores are the most common method of reproducing molds.
• Some molds can also produce sexual spores. These molds are called “perfect” and can be classified as Oomycetes, Zygomycetes, Ascomycetes, Basidiomycetes, or Oomycetes, if they are septate. This is in contrast to the “imperfect” molds, which are typically septate and have only asexual spores.

(a) Asexual Spores  of Molds

• Asexual spores of molds are abundant and are small, and light. They are also resistant to drying.
• They can be easily spread throughout the air and set off new mold thallus when conditions are favorable.
• These are the three main types of asexual spores.
  • Conidia (singular Conidium): Conidia, also known as single conidium, are taken from fertile hyphae called Conidiophores. They are usually in the open, which means they are not enclosed in any container.
  • Arthrospores or oidia: These are formed in a sporangium (pluralsporangia), or at the tip a fertile hypha, called the sporangiophore.
  • Sporangiospores – Sporangiospores form from the fragmentation of a Hypha. The cells of the Hypha then become arthrospores.
  • The fourth type of asexual spore is the chlamydospore. It’s formed by many types of molds. A cell in the mycelium that stores food reserves, swells and forms thicker walls than the surrounding cells. The chlamydospore (or resting cell) can survive unfavorable conditions more than normal mold mycelium can. Later, under favorable conditions it can turn into a new mold.
• It is possible to identify genera and species of molds by studying the morphology of the sexual spores.
• Sporangiospores can vary in size, shape, color, and even color. These conidia can be either smooth or roughened, one-, two, or many-celled.
• The appearance of fertile hyphae on the molds and the asexual spores on them is also helpful in identifying the molds.
• When sporangiospores form, it is important to note whether they are branched or simple, as well as the type and size of the branching.
• The columella, the swollen tip on the sporangiospores that usually extends into the sporangium and is usually swollen, takes shapes similar to species of mold.
• Conidia can be produced singly or in spore heads with different arrangements and complexity.
• It is often enough to identify the genus by looking at the general appearance and shape of the spore head.
• Some molds have conidia that are in chains. They are squeezed one at a time from a special cell called a sterigma or phialide at the tip. Some molds may have conidia in irregular mass, which are cut from the conidiophore’s tip without any sterigmata. These conidia masses can be either loosely packed, tightly packed, or even enslimed.
• Some molds’ conidia bud from the conidiophore, and then continue to multiply by budding; it looks yeastlike.

(b) Sexual Spores  of Molds

These spore-producing molds are classified based on the manner of formation of these spores and the types produced.

• Oomycetes are the nonseptate molds (Phycomycetes), that produce oospores. These molds are mostly aquatic, but include several important plant pathogens such as the “downy mildews”, which cause late blight and buckeye-rot in tomatoes.
• The union of a small male and large female gamete creates the oospores.
• Zygomycetes are formed by the union of the tips from two hyphae, which can often look similar and may be from the same mycelium or different mycelia.
• Both zygospores, as well as oospores, are protected by a strong wall that can withstand drying for extended periods.
• The Ascomycetes (septate), are formed from sexual spores called as ascospores. These spores are made from cells of the same mycelium, or two different mycelia.
• Ascospores are the result of cell division following conjugation. They are usually found in an ascus (or sac) with eight spores each ascus.
• Asci can be single or grouped together in a covering called an “ascocarp”, formed by interconnecting adjacent hyphae and branching.
• Basidiomycetes are mushrooms that can plant rusts, smuts and other spores.

Cultural Characteristics of Molds

• It is often enough to identify the class and order of a food born mold by its appearance.
• Some molds are loose, while others are more compact.
• Some appear velvety on their upper surfaces, while others are dry and powdery and some wet and gelatinous.
• Some molds have a limited size while others are smaller due to the container or food they are being used in.
• There are distinct zones of growth in the Thallus that distinguish some molds, such as Aspergillus niger.
• The mycelium has pigments that are red, purple, yellow, brown, gray, black, etc–are characteristic, as are the pigments of masses of asexual spores; green, blue-green, yellow, orange, pink, lavender, brown, gray, black, etc.
• The reverse side of molds on agar plates can be quite striking. This is similar to the opalescent or greenish-black Cladosporium underside.

Physiological Characteristics of Molds

• Moisture requirements: Most molds need less moisture than most yeasts or bacteria. It is possible to estimate the limiting moisture content of food items for mold growth. Accordingly, it is believed that foods containing less than 14-15 percent total moisture will inhibit or delay mold growth.
• Temperature requirements: Most molds can grow at normal temperatures. Most molds grow best at 25-30 C. However, some molds can grow at 35-37 C or higher, such as Aspergillus species. Some molds also thrive at higher temperatures. Many molds are psychrotrophic, meaning they can grow well at low temperatures and others slow down at lower temperatures. Some molds have been found to grow at temperatures as low as -5C to -10C. Some plants are thermophilic, meaning they can tolerate high temperatures.
• The oxygen and pH requirements: Molds are aerobic. This means that they need oxygen to grow. While most molds can grow in a range of hydrogen-ion levels (pH 2-8), the majority prefer an acid pH.
• Food requirements: Most molds can use a variety of foods from simple to complex. Many common molds have a wide range of hydrolytic enzymes. Some are grown for their amylases and pectinases as well as proteinases and lipases.
• Inhibitors: Some molds produce inhibitors. These compounds are harmful to other organisms. Mycostatic compounds called Certai chemical substances are those that inhibit the growth of molds (sorbic, propionates and acetates are some examples), or they are specifically fungicidal which kill molds. Molds are slower than bacteria and yeasts in initiating growth. This means that molds often lose out to other organisms when the conditions are right. However, mold growth can be quite rapid once it is underway.

Classification And Identification Of Molds

Molds are plants belonging to the kingdom Myceteae. They lack chlorophyll and have no roots, stems, or leaves. They are part of the Eumycetes or true fungi and can be subdivided further into subdivisions, classes, orders, and families as well as genera.

These criteria are used primarily for the identification and differentiation of molds.

• Hyphae septate, or nonseptate
• Mycelium is clear or dark (smoky)
• Mycelium is colorless or colored
• How many sexual spores are made and which type of spores they are: oospores or zygospores.
• There are three types of asexual yeast spores: conidia, sporangiospores, and arthrospores (oidia).
• Characteristics of the sporehead
  • Sporangia: Size, color, shape and location
  • Spore heads bearing conidia include single conidia, conidia in chains, budding conidia or masses; shape and arrangement of sterigmata, phialides, and gumming conidia together
• Conidiophore appearance: sporangiophores and conidiophores can be either branched or simple. Size and shape of the columella at the tip of sporangiophores. Conidiophores can also be single or in bundles.
• Microscopic view of the asexualspores, particularly of conidia, including shape, size and color.
• Special structures (or spores), may be present in stolons and rhizoids, foot cell, apophysis, and chlamydospores.

Samson et al. have provided a great introduction to food-borne fungi. (1984). The text includes illustrations of many food-borne molds, photomicrographs of actual molds, photos of molds on agar surface, and a key for identification.

Industrial Importance of Molds

Several molds of industrial importance are outlined by genus. The corresponding Figures show the typical morphological structures.

• Mucor: Mucor can be involved in spoilage and manufacturing of other foods. M. racemosus is a widely-distributed species. M. rouxii can be used in the “Amylo” process to saccharify starch. Mucors are used to ripen certain chesses (e.g. Gammelost), and they are also used in certain Oriental food recipes.
• Zygorrhynchus – These soil molds look similar to Mucor, except that their zygospore suspensors have a markedly different size.
• Rhizopus: Rhizopus, also known as the bread mold, is very common. It is responsible for spoilage of many foods, including fruits, vegetables and bread.

• Absidia: Akin to Rhizopus except that the sporangia look smaller and more pear-shaped.
• Thamnidium: Thamnidium is found in meat during chilling storage. It causes “whiskers”.
• Aspergillus: It is a very common species. Some are responsible for spoilage and others are helpful in certain food preparation. Raper and Fennell (1965), list 18 groups of aspergilli, and recognize 132 species. However, only a few species will be discussed here. Food spoilage is often caused by the A. glaucus, which includes A. repens. Molds can grow in foods with low moisture content and high sugar and salt concentrations. This group has conidia that are a shade of green. Ascospores can be found in asci with yellow-reddish perithecia. These molds are included in the Ascomycetes, and the genus Eurotium. This is a name reserved to members with a perfect (sexual) stage. A. niger is the leading species in the A. niger group and could be used to make food. The spore-bearing heads of A. niger are large, densely packed and globular. They can be black, brownish, or purple-brown. The conidia are rough and have bands of pigment. Many strains are blessed with sclerotia. They can be colored from blackish to grayish to buff. A few strains are used in the production of citric acid and gluconic acid, as well as in enzyme preparations. A. flavus oryzae molds are important for the marking of certain Oriental foods as well as the production of enzymes. However, these molds can also cause food spoilage. Conidia can give spore heads a variety of yellow-green shades, and dark sclerotia might be formed.

• Penicillium: Penicillium is another genus, which is common in occurrence and is important in food. There are many species in the genus. The subgroups and groups of the genus are divided. Based on the number of spore-bearing heads or penicilli (little brush), the genus can be divided into large groups. Verticillata are heads that contain three or more elements. These elements include sterigmata (subbranches), metulae and branches. P. expansum, the blue green-spored mold, causes soft-rots in fruits. P. digitatum has olive- or yellowish green conidia and causes soft rots of citrus fruits. P. camemberti has grayish conidia that aid in Camembert cheese ripening. P. roqueforti has bluish-green coneidia which aids in the ripening blue cheeses like Roquefort. Some species can form asci in cleistothecia with ascospores, while others have sclerotia.
• Trichothecium: Trichothecium is the common species of T. roseum. This pink mold grows on wood, paper and fruits like apples and peaches as well as vegetables such as cucumbers, cantaloupes, and even paper. The clusters of conidia, which are two-celled, can be easily identified by the short, erect conidiophores. The conidia have a nipple-like projection at their point of attachment. This is where the smaller of the two conidia cells is located.
• Geotrichum (Oospora, Oidium): Some writers include this genus with yeastlike fungi and others with molds. The growth may appear as a hardened, felt-like mass at first, but then becomes soft and creamy. Geotrichum candidum, also known as the “dairy mould”, produces white-to-cream-colored growth. Hyphae are septate, and are often dichotomously branching. Asexual spores can be described as arthrospores (oidia) and may appear rectangular if they are submerged hyphae or oval if they are aerial hyphae.
• Neurospora (Monilia): This genus was originally called Neurospora, but it has since been referred to under many names due to confusion regarding its classification. Most mycologists believe that the perfect molds (producing sexualspores) should be considered and the genus Neurospora should be named. Neurospora Neurospora (Monilia). (Monilia), sitophila is the most important species of foodstuffs. Its pink, loose-textured growth frequently occurs on bread. It can also be found on sugarcane bagasse, and many other foods. Rarely is the perfect stage, also known as ascosporogenous.

• Sporotrichum: Sporotrichum is a saprophytic species that grows on chilled meats and causes “white spots.”
• Botrytis: B. cinerea is an important species in food. It can cause grape disease, but it may also grow saprophytically on other foods.
• Cephalosporin:  C. acremonium, a very common species.
• Trichoderma: T.viride is a very common species. The mature mold plant’s color is bright green due to the gluing of the green conidia balls together. Sterile tufts with hyphae stick well above the conidiophores.
• Scopulariopsis: S. Brevicaulis, a common species. Penicillium may be mistaken for this genus, as both have brushlike penicilli, chains of spores, and cut-off sterigmata. However, Scopulariopsis conidia are never green. Scopulariopsis can have conidiophores that are branched or not branched, simple or complex and sometimes with irregular branching. There are many types of spore-bearing heads, ranging from complex and branching systems with penicilli to single stems arising from short branches from aerial hyphae. Microscopically, the spores have a distinctive appearance. They are usually yellowish-brown and not green. Colonies are brownish-colored and cottony.
• Pullularia: Hyaline conidia, or blastospores from preexisting cells), borne as lateral buds in all parts of mycelium. When young, colonies are yeast-like and pale. As they age, they become mycelial and darker and more leathery. P. pullulans, a common species.

• Cladosporium: C. herbarum, a prominent species. These dark molds can cause “black spot” in a variety of foods and cellar walls. C. herbarum colonies are limited in growth, thick, velvety and olive-to-gray-green. The reverse side of the plant is strikingly opalescent and greenish-black.
• Helminthosporium: Species in this genus can be plant pathogens for most of their lives, but they may also grow saprophytically on vegetables.
• Alternaria: Common causes of food spoilage are molds from this genus. A. citri, A. tenuis and A. brassicae all occur in common species. Although the mass of mycelium is usually dark gray-green, hyphae can often appear almost colorless when viewed under a microscope. The conidiophore is a chain that contains the brown, many-celled conidia.
• Stemphylium: Stemphylium is also a common genus. The conidia are multicellular and dark in color, but they have less cross walls than Alternaria’s and are round at both ends.

• Fusarium: Fusarium is a genus of molds that often grows on food. It is difficult to identify the species and their growth patterns are variable.
• Endomyces: Endomyces is a yeastlike fungi that forms mycelium and arthrospores. Some species can rot fruit.
• Monascus: The colonies of M. purpureus are reddish-purple or purple and thin. It is found in dairy products as well as on Chinese red rice (angkhak).
• Sclerotinia: Some species cause rots of vegetables and fruits, where they are present in the conidial stage. The conidia of the lemon shape are found in chains with a plug that separates conidia.