NISIN RECOVERY FROM BY-PRODUCTS CONTAINING
FERMENTABLE SUGARS FROM A BREWERY INDUSTRY
Pablo José Rosales Pineda
pjrosales@outlook.com
Chemical engineer
Advised by: Eng. Álvaro de León Marizuya
ABSTRACT
An assessment was made with four
different recovery and purification
protocols, establishing as comparison
parameters:the yield, the total
recovered amount and the physic-
chemical properties of the culture
media containing fermentable sugars
derived as by-products from a brewery
industry.
The selected recovery protocols
integrate exclusively the use of food-
grade reagents. The brewery industry
has a wide selection of sources
containing sugars (extract): 5º diluted
wort, spent grain pressed water, hot
sludge (trub) liquid extract, yeast beer
extract and the Lauter’s last runnings,
as the five main culture media for the
Nisin insertion for production
directions. The antimicrobial activity
was verified with the Kirby-Bauer’s
methodology of solid agar diffusion.
(KB-SAD).
This research revealed that there is a
significant difference between every
evaluated protocol and defined the
Ammonium Sulphate in wort as the
best recovery protocol, getting the
maximum recovered Nisin with 80.07%
± 1.013 % yield. The Ethanol + Sodium
Chloride protocol got the highest
yields over the rest of protocols in 5
different culture mediums, with an
average recovery yield of 61.35% ±
1.013 %.
The Lauter’s last runnings and the
spent grain pressed watersamples
were found as the culture medias with
the best conditions for the insertion of
LAB (lactic acid bacteria) for the Nisin
production, keepingthe recuperation
yield in the highest rate.
Keywords:
Nisin; Lactic acid bacteria (LAB); Bacteriocins;
Bacteriocidal activity;Beer; Fermentation;
Preservantives; GRAS.
INTRODUCTION
Recently, consumers have been
increasingly concerned about the
possibleadverse health effects of
chemical additives on food, boosting
considerably the demand for organic
foods and natural additives and
preservatives.
The implementation of Nisin represents
a potential alternative as a natural
preservative, due to its inhibitory
activity against a wide range of Gram-
Positive bacteria, including their
sporulated forms.
Nisin is a peptide with a bactericidal
activity, produced by certain stripes of
Lactococcus lactis subp. lactis in
optimal conditions of fermentation in
rich and nutritive culture media, and
enhancers for the bacteriocins
production.
Several analogue protocols base don
Nisina researches have shown its
stability in high temperatures,
especially when there is a media with
acid conditions, enduring its efficacy
thru sterilization processes as boiling
and pasteurization process.
There are no reports on negative
interactions with the food’s original
organoleptic and physic-chemical
characteristics, nor toxic
consequences for human
consumption. Nisin is disintegrated
naturally into the human intestine as a
protein.
The toughest constraint on Nisin
production protocols is
theimplementation ofstrong and toxic
reagents employed in the actual
recovery protocolsapplied in the food
industry, low yield, high difficulty
application and expensive operation
costs.
The objective in this research is to
keep the line on the exclusively usage
of food-grade reagents,in order to not
to interfere in the original
characteristics of the product.
The application of Nisina in the food
industry is based on its antimicrobial
properties, looking to increase the
product’s shelf life, to prevent and to
eliminate any possible infections in the
production processes, and to support
to decrease the lazy times and costs in
sterilization processes, functioning as a
double inhibitory backup.
CONCLUSIONS
1. The yeast beer extract is the by-
product with the lowest chances as
its implementation as a culture
media.
2. The Lauter’s last runnings and the
spent grains pressed water are the
best culture media, having the most
favourable media for the
applications as matrix media for
Nisiin producing bacteria (LAB).
3. The yeast beer extract is the culture
with the lowest chances for its
posible application as a culture
media, due to its high alcohol
content, turbidity, BU, polyphenols
and extract. These factors hinder
dramatically the performance of the
LAB producing Nisin.
4. The Ammonium Sulphate is the
protocol with the highest Nisin
recuperation yield with 80.07% in
wort, precipitating 2.00 mg of pure
Nisin.
5. The ethanol + sodium chlorine got
the next 3 highest Nisin recuperation
yields in every culture media
evaluated with78.05%, 72.21% and
68.63%.
6. El Calcium Hydroxide flocculation
protocol was the only procedure
which no hibition zone was
observed, nor was any active Nisin
milligram recovered.
RECOMMENDATIONS AND FUTURE
PROSPECTS
1. Simultaneous filtrations may
improve the extraction and filtration
time and helpstoavoid possible
contaminations due to its dense
nature.
2. A key element in this research was
to establish the method described in
Taylor and Müller-Auffermann’s
protocols for the antimicrobial
activity determination: difussion in
liquid culture media and letting it to
solidify. Discard the Weber’s method
of spreading a thin lay of the
bacteria over the solid agar and no
into it.

3. In order to reach the highest
recovery yield combined with the
lowest contaminants content, the
recovered samples can pass throuh
a spray-drying procces, where the
solvent evaporates quickly from the
droplet, eliminates the moisture,
resulting in a solid-powder
presentation of the product. This
won’t interact with the original
organoleptic propierties for better
food-industry applications.
4. Rotoevaporation and recuperation of
the alcoholused in the recovery
protocols would let the recirculation
the condensate for reuse, optimizing
and increasing the profitability of the
process.
5. The Nisin has a potential application
in the brewery´s fermentation
procces, which would represent a
significant advantage in favour of
brewing yeasts.
6. The implementation of Nisin in the
brewery’s pasteurization process
may lead intosavingenergy, costs
and eliminating the risk of the
product’s damage caused by high
temperatures by decreasing the
current operating times and
temperatures in pasteurization
tunnels.
for Brewing and Food Quality, Technical
University of Munich (TUM). Freising, Germany.
2014b.
5. PARADA, J. L. / CARON, C. R. / MEDEIROS, A.
B. / SOCCOL, C. R. Bacteriocins from lactic acid
bacteria: purification, properties and use as bio-
preservatives. Unidadede Biotecnologia
Industrial: Divisãode Bioprocessos e
Biotecnologia; Setor de Tecnologia;
Universidade Federal do Paraná.Curitiba,
Paraná, Brasil. 2007.
6. PINGITORE, E. V. / SALVUCCI, E. / SESMA, F.
/ NADER-MACIAS, M. E. Different strategies for
purification of antimicrobial peptides from lactic
acid bacteria (LAB). Centro de Referencia para
Lactobacilos CERELA-CONICET. Tucumán,
Argentina. 2007.
7. TAYLOR, T. M. / DAVIDSON, P. M. / ZHONG,
Q. Extraction of Nisin from a 2.5 % commercial
Nisin product using methanol and ethanol
solutions. Department of Food Science and
Technology, Tennessee University. Knoxville,
Tennessee, United States of America. 2007.
THANKFUL TO
To God, to my parents, my family,
close friends and all the people that
made this possible.
Pablo José Rosales Pineda
Chemical Engineer
San Carlos’ de Guatemala University

REFERENCES

1. BURIANEK, L.L. / YOUSEF, A. E. Solvent

extraction of bacteriocins from liquid cultures.
Department of Food Science and Thecnology,
The Ohio State University, Columbus, Ohio,
United States of America. 2000.
2. CHEESEMAN, G. C. / BERRIDGE, N.An
improved method of preparing Nisin. National
Institute for Research in Dairying, Reading
University. Reading, England. 1956.
3. MÜLLER-AUFFERMANN, K. / GRIJALVA, F. /
JACOB, F. / HUTZLER, M. Nisin and its usage
in breweries: a review and discussion. Research
Centre Weihenstephan for Brewing and Food
Quality, Technical University of Munich (TUM),
Freising, Germany. 2014a.
4. MÜLLER-AUFFERMANN, K. / GRIJALVA, F. /
JACOB, F. / HUTZLER, M. Nisin-producing
microorganisms and their implementation in
brewers’ wort. Research Centre Weihenstephan