Department of Chemical Engineering

University of San Carlos Technological Center

Nasipit, Talamban, Cebu City

ChE 426 N

Introduction to Biotechnology

A Critique on a

Lemon Juice Clarification Using Fungal Pectinolytic Enzymes Coupled to Membrane

Ultrafiltration

Submitted to

Dr. Camila Flor Y. Lobarbio

by

Carl John Louie G. Navalta

March 31, 2016

Introduction

There are a lot of reasons why juicing fruits and vegetables are incorporated by nutritionists
in every health program (Mercola, 2011). All nutrients present in fruits and vegetables are absorb
effectively in our body through juices. But on the contrary, the American Journal of Public Health
proposed that the Hunger-Free Kids Act of 2010 in the United States has eliminated all of the fruit
juices because it has been linked to childhood obesity (Wojcicki, 2012) and the American Cancer
Society claims that there are no convincing scientific evidences that extracted juices are healthier
than the whole fruit or vegetable.

Most fruit and vegetable juices are cloudy in appearance after extraction. This is due to the
pulp particles suspended and the naturally occurring substance called pectin (Robards et al., 1999).
In unripe fruit, pectin is attached to the cell walls through cellulose microfibrils. It is insoluble and
the liquid within the cells remains as fluid. This makes the cell wall rigid. When the fruit or
vegetable ripes, the pectin is altered by naturally-occurring enzymes. The pectin long chain is
broken-down, and this results to pectin becoming more soluble and its grip to the cell wall is
loosened, and the plant tissues soften. This suggests that the ripe fruits or vegetables are easier to
press and extract the juices, where some of the pectin molecules are released into the juice making
it appear viscous (National Centre for Biotechnology Education NCBU, 2000).

A clarification step is necessary in order to reduce the turbidity of juices. This may involve
the use of pectinases and other enzymes in fruit juice to overcome this problem. Pectin coats the
proteins in the fruit in suspension however, in acidic conditions pectin is negatively charged and
it repels with the protein. With the use of pectinases, it will degrade the pectin molecules and
causes aggregation of cloud particles which then can be precipitated out from the juice. Fungal
enzymes that exhibit pectinolytic activity are also available. These fungal enzymes can exhibit
such activity even in very acidic juices. Clarification with the use of these enzymes can be achieved
in 6 hours and without the need for preservatives (Ray and Ward, 2008).

Fungal enzyme clarification can be coupled with membrane filtration processes,

ultrafiltration process (UF), which are increasingly used in juices and beverage industries. This is
because of the fact that membrane processes can be carried out in a room temperature and
minimizes the loss of aromatic volatile substances. But this process can face a major hindrance.

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The filtration performance decreases with increasing filtration time due to membrane fouling
which refers to the blockage of membrane pores by particulates and compounds present in the
juice (Abdelrasoul et al., 2013). This can be remedied by pre-treatment of the feed in order to
control colloidal, organic, and biological fouling and as well as scaling. In the article, the pre-
treatment was the use of pectinoytic enzymes produced by Penicillium occitanis. This pre-
treatment process is called depectinization, where pectic enyzmes hydrolyze pectin and reduce the
viscosity of the juice and can be easily filtered (Sahin and Bayindirli, 1993).

Summary of the Study

The methods carried out were (a) pectinase production, (b) enzyme assays, (c) pH and
temperature effects, (d) enzymatic treatment, (e) UF treatment, (f) physic-chemical analysis, (g)
microbiological analysis and (h) statistical analysis.

Pectinase used were produced from a cellulolytic fungus, a species of Penicillium that was
isolated from soil. Different carbon sources were used and the culture was incubated at 30oC for 5
days, resulting into orange peels that contribute much to the pectinase activity. The broths were
clarified through centrifugation for 15 minutes and filtered to remove the mycelia. The filtrates
were used as crude enzyme extract.

The activity of the enzyme produced was determined using the dinitrosalicylic acid reagent
DNS assay (Miller, 1959). This measures the release of reducing groups. A mixture of citrus
pectin, citrate-phosphate buffer, crude enzyme extract was incubated at 60oC for 10 minutes, and
the enzymatic activity (U) was determined in mol of galacturonic acid released per minute.

The optimum pH for pectinase activity was studied over a pH range of 2.0-9.0. The enzyme
was kept at 4oC in different buffers for 24 hours to determine the pH stability, and under assay
conditions, the residual pectinase activity was determined. The enzyme activity is at peak at pH 6.
The activity was also studied over temperature range of 20-70oC to determine the effect of
temperature on the enzyme activity, and thermal stability was determined after different incubation
times, 0-90 minutes at 20-50oC. It was found and reported that at 60oC, enzyme activity is
optimum.

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 Aliquots of juice were distributed into glass containers for each of enzymatic treatments.
The lemon juice was treated with pectinase at various enzyme concentrations, temperature and
times. A pre-filtration was performed before ultrafiltration to remove most of the pectin-protein
flocs formed during enzyme treatment. During UF treatment, lemon juice was circulated through
the membrane unit using a peristaltic pump. The feed temperature is 20oC, feed flow is 0.9 L/min
and transmembrane pressure is 0.3 MPa.

Physico-chemical properties such as dry matter, protein, ash and total sugar content, total
phenolics, turbidity, viscosity, clarity and color, and pH were determined. Dry matter, protein and
ash was determined using AOAC methods. Total phenolics were determined using Folin-
Ciocalteau reagent. Total sugar was measured after acid hydrolysis at 100oC. The turbidity of the
juice was determined using a turbidimeter. The viscosity of the juice was determined at 25oC with
a Stress Tech Rheologica Rheometer at a constant shear rate, and shows that at pectinase
concentration of 600U/L, a temperature of 30oC, and treatment duration of 90 minutes, the juice
has low viscosity. Clarity and color was determined using a UV-vis spectrophotometer. The pH
level of the juice was determined using a pH meter.

The total plate counts, formation of molds and coliforms, Enterobacteriaceae and
Staphylococcus counts of all samples were determined at zero time and during storage at room
temperature during 3 months. The clarified juice did not show significant development of molds,
coliforms and bacteria.

Analytical values were determined using three independent determinations. Values of

different parameters were reported as the meanstandard deviation. To establish significance of
difference between the different treated lemon juices, students t-test at the level of P<0.05 was
applied to the data.

Evaluation

The use of P. occitanis is a good choice in producing pectinase. P. occitanis (PO16) is a

mutant that produces a broad spectrum of polysaccharide-hydrolising enzymes (Jain et al., 1990).
In the extraction of lemon juice stated in the materials section of the study, the researchers did not

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include the sanitations that should be carried out in the extraction process which is very important
so that the end product is not contaminated with bad microorganisms.

One good thing about this paper is that it includes the physico-chemical composition of
lemon juice. These compositions are basis on describing the color and appearance of the juice. It
also presents the amount of pectin present in lemon juice, where according to NCBU, is the reason
why juices, particularly lemon juice has a turbid or viscous appearance. The study also includes
the study on basic parameters that affect the treatment of lemon juice by enzyme and UF.

The researchers present the results of the experiment through graphs which the reader can
base upon so that in the results and discussions, they will be able to refer on these graphs and
validate the researchers explanations and discussions. Although the plots presented include
activity percentages which are not very clear, as to which factor is the enzyme activity relative to.
The researchers report that the optimum temperature for enzyme activity is 60oC, although
referring to figure 3, the peak of enzyme activity is at, more or less, 55oC. They also conclude that
through the partial characterization of pectinase activity, the enzyme is capable for juice
clarification, but did not site any reference that could back up this conclusion.

The researchers cite many references in their paper which can help the readers to validate
the facts stated. The citations can also be used as an additional information for the readers.

The objectives of the study are not stated in the paper, which can help in drawing the
readers attention and make them interested in the study. And the statement of the objectives will
help in construction of conclusion. The paper has many errors on grammars and use of punctuation
marks.

Conclusion

The combination of fungal enzyme treatment and membrane filtration is an effective way
to degrade pectin molecules present and through UF, these conglomeration of pectin molecules
are easily removed from the juice.

Addition of pectinolytic enzyme to lemon juice before ultrafiltration will prevent

membrane fouling, and will not decrease the filter efficiency. This pre-treatment also helps in the

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characterization of the enzymes that will be used on clarifying the lemon juice. Enzyme activity is
optimized through investigation of the different parameters; temperature, pH and treatment
duration. Enzyme concentration is also very important to study to be able to effectively reduce the
viscosity of the lemon juice.

References

Abdelrasoul, A., Doan, H. Lohi, A. 2013. Fouling in Membrane Filtration and Remediation
Methods. Retrieved on March 23, 2016 from http://cdn.intechopen.com/pdfs-wm/45134.pdf

Jain, S., Durand, H., Tiraby, G. 1990. Production of Extracellular Pectinase Enzymes By a Mutant
(PO16) of Penicillium occitanis. Applied Microbiology and Biotechnology. Vol. 34, Issue 3. pp
308-312

Mercola, J. 2011. Benefits of Juicing: Your Keys to Radiant Health. Retrieved on March 23, 2016
from http://articles.mercola.com/sites/articles/archive/2011/11/13/benefits-of-juicing.aspx

Miller, G.L. 1959. Use of Dinitrosalicyclic Acid Reagent for Determination of Reducing Sugar.
Analytical Chemistry. ACS Publications. Sixteenth Street N.W. Washington DC. pp 426-428

National Centre for Biotechnology Education. 2000. Enzymes in Fruit Juice Production. Retrieved
on March 23, 2016 from http://www.ncbe.reading.ac.uk/ncbe/protocols/inajam/pdf/jam01.pdf

Ray, R., Ward, O. 2008. Microbial Biotechnology in Horticulture. Volume 3. CRC Press. Broken
Sound Parkway NW, Suite 300, Boca Raton, FL

Robards, K., Prenzler, P., Tucker, G., Swatsitang, P., Glover, W. 1999. Phenolic Compounds and
Their Role in Oxidative Processes in Fruits. Food Chemistry. Vol. 66, Issue 4. pp 401-436

Sahin, S., Bayindirli, L. 1993. The Effect of Depectinization and Clarification on the Filtration of
Sour Cherry Juice. Journal of Food Engineering. Vol. 19, Issue 3. pp 237-245

Wojcicki, Janet M.; Heyman, Melvin B. 2012. Reducing Childhood Obesity by Eliminating 100%
Fruit Juice. American Journal of Public Health