CPB 30103 1

BIOCHEMICAL ENGINEERING

Experiment 5
Food Grade Red Pigment Production by Penicillium sp.

INTRODUCTION

Biochemical engineering is a branch of chemical engineering that deal with the

design and construction of unit processes and unit operations that involve biological
organisms or molecules. It includes cell or microbial culture technique and separation
processes for bio-products production, food processing and biological waste treatment.
Biochemical engineers apply the principle of biology, chemistry and engineering to
produce useful products such as biopharmaceuticals, biofuels, biopolymers, pigments and
industrial enzymes. There exists a relationship between biological sciences, processes and
chemical engineering that amalgamate to form a biochemical process industry. The
interdisciplinary interaction taking place today between microbiologists, biochemists and
chemical engineers is explained by table below.

Biological sciences Processes Chemical Engineer

 Fermentation  Culture choice  Reactor design
technology
 Genetics  Mass culture  Process control
 Biochemistry  Cell response  Unit operations
 Physiology  Process operation  Energy and material
utilization
 Chemistry  Product Recovery

THEORY

Pigments are compounds with characteristics of importance to many industries

such as food and beverage industries where they are used as additives, colour intensifiers,
antioxidant and so on. In fact, pigments come in huge variety of colours and some of
them are soluble in water. The negative perception of synthetic food pigments by modern
consumers have arisen a strong interest in natural colouring. Furthermore, the increased
in the concern of synthetic pigments such as azorubin and tartrazin which are potentially
toxic and allergic has led to an increased demand for natural pigments. Due to those
reasons, many companies have decided to utilize natural pigment mainly from plant and
animal sources, but these additives have numerous drawbacks including instability and
low water solubility. Furthermore they are often not available throughout the year.
Therefore, pigments from microorganisms (bacteria, yeasts and fungi) are of industrial
interest because they are often more stable and soluble than those from plant or animal
sources. Microorganisms can grow rapidly which can lead to high productivity and
availability throughout the year.
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BIOCHEMICAL ENGINEERING

Fungi can be single celled or very complex multicellular organisms. They are
found in just about any habitat but most live on the land, mainly in soil or on plant
material rather than in sea or fresh water. A group called the decomposers grow in the soil
or on dead plant matter where they play an important role in the cycling of carbon and
other elements. Some are parasites of plants causing diseases such as mildews, rusts,
scabs or canker. In crops fungal diseases can lead to significant monetary loss for the
farmer. A very small number of fungi cause diseases in animals. In humans these include
skin diseases such as athletes’ foot, ringworm and thrush. Filamentous fungi are a
significant natural-pigments source, because the fungal pigments could easily be
produced in high yields via cultivation technology especially in submerged fermentation
system. In fact, many species of Penicillium, such as .P herquei, P. persicinum, P. fagi
and so forth, have been explored as the potential natural pigments producers. A
commercial food colorant called Arpink Red™, which is produced by a variety of P.
oxalicum is already available in the market.

For an industrial fermentation process fermentation medium and fermentation

process condition plays an critical role because they effect the formation, concentration
and yield of a particular fermentation end product thus effecting the overall process
economics therefore it is important to consider the optimization of fermentation medium
and process conditions in order to maximize the profits from fermentation process. There
are many challenges associated with optimization of fermentation process, it is laborious,
expensive, open ended and time consuming process involving many experiments. In
bioprocess industry it is often needs to conduct optimization experiments because new
mutants and strains are continuously being introduced. In fermentation process
optimization different combinations and sequence of process conditions and medium
components are needs to be investigated to determine the growth condition that produces
the biomass with the physiological state best constituted for product formation.

LABORATORY PROCEDURE

Apparatus

1. 250 ml Erlenmeyer flask containing 100 ml of yeast extract sucrose broth (pH6)
2. Micropipette and sterile blue tips
3. Spore suspension of Penicillium sp.
4. Whatman No1 filter paper
5. Filter funnel with 250 ml Erlenmeyer flask
6. Autoclave
7. Weighing balance
8. 60 °C oven
9. Spectrophotometer and cuvette
10. Beakers
11. pH meter
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BIOCHEMICAL ENGINEERING

Procedure
Waste and Spills:
All of the micropipette tips used in this experiment CANNOT be discarded via drain
disposal with running tap water. Please discard in a beaker. Some of the bacteria are
pathogenic, please follow the safety instructions when working with microorganisms.

***REMINDER: ASEPTIC TECHNIQUE

A. Inoculation of fungal culture (Week 8)

a) Transfer 1 ml of the fungal spore suspension aseptically into yeast extract
sucrose medium (sucrose 40 g/L, yeast extract 20 g/L and magnesium
sulphate 0.5 g/L; pH 6).
b) Incubate the flask cultures for 14 days at room temperature under static
condition.
c) On daily basis, shake to mix the cultures.

B. Observation of fungal culture (Week 10)

a) Examine the 7 days-old culture of Penicillium sp. on potato dextrose agar
plate. Describe the following characteristics of the fungal culture
 Diameter of colony (Get 3 measurements and report your data as
mean ± standard deviation)
 Color of the colony
 Color of reverse colony
 Color of spores
 Form
 Margin
 Pigmentation on the agar
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BIOCHEMICAL ENGINEERING

C. Measurement of pigment production and dry biomass (Week 10)

a) After the incubation period, filter the culture with Whatman No. 1 filter
paper (see below). Pre-weigh your filter paper.

b) Check the absorbance of the filtrate at 500 nm with spectrophotometer.

Express the pigment yield as optical density unit.
c) Dry the filter paper (with known weight) with fungal biomass overnight at
60 °C. Check the weight of the dry biomass by weighing balance.

QUESTIONS

1. Discuss industrial applications of Penicillium sp.

2. Explain the concept of “Microbial cell factories”.