Yali Friedman
November 25th, 1996
Yali Friedman
November 25th, 1996
The single-most important development permitting the formation of civilization was the
ability to produce and store large quantities of food. Hunter-gatherer societies lived
from day to day either starving or gorging themselves based upon the amount of food they
could find in a day. When it became possible for one person to produce more food than they
needed, time from gathering food could be apportioned to culture and science. Following
this trend, it became beneficial to be able to store as much food as possible in order to
minimize the amount of time spent gathering that food. Food storage has always been at
odds with food spoilage. Some of the earliest evidence of food preservation comes from the
Post-Glacial era, from 15,000 to 10,000 BC. Improved weapons and strategies allowed the
killing of large animals such as mammoths and reindeer. This improved efficiency allowed
the collection of more meat than could be eaten in a day and this meat was either frozen
in pits or stored in the colder parts of caves. The first use of biological methods was
from 6000 to 1000BC when fermentation was used to produce beer, bread, wine, vinegar,
yogurt, cheese and butter. Spices and herbs were also used. In 1864, Louis Pasteur proved
that microorganisms in foods were the cause of food spoilage, that heat treatment of food
killed these microbes and that sealed containers helped to preserve food by preventing
recontamination from atmospheric air. A major development in the distribution and storage
of foods came in 1940 with the availability of low-cost home refrigerators and freezers.
This, combined with the development of affordable refrigeration and freezing facilities
for processing, storage and transportation of foods allowed refrigerated and frozen foods
to become very important commercial items. Other developments included the artificial
drying of fruits, vegetables and liquids, vacuum packaging, ionizing radiation, and
chemical preservatives. One significant development in biological controls was the
discovery of nisin, a peptide metabolite of food-grade starter culture bacteria. Nisin was
found to have antimicrobial properties against many spoilage and pathogenic bacteria[1].
With the variety of different techniques that can be used to sterilize food, it might
seem that there is no need for new developments. Many consumers today are concerned about
the synthetic "chemicals" used as preservatives in food, and there is a
resulting trend towards less processed food.Alternatives include vacuum packing and
refrigeration of foods. These untreated foods can harbour dangerous pathogens which can
multiply under refrigeration and without oxygen (psychotrophs). Treatments like ionizing
radiation can destroy pathogens non-chemically, but may affect taste and do not protect
food against post-treatment contamination. A solution to this dilemma is the use of
antimicrobial metabolites of fermentative microorganisms.
In the United States, antimicrobial food preservatives fall under four groups: Those that
are not defined by law (natural organic acids and their salts, spices, oils, and wood
smoke); Those that are Generally Regarded As Safe (GRAS); Those that have been proven to
be safe and approved by regulatory agencies, and; Those that are not currently in use and
where proof of safety has yet to be established. The greatest amount of concern seems to
be centred around newer chemicals, for which long-term affects are not determined. Many
antimicrobial chemicals have been in use for a long time without any known adverse
affects. These include natural components found in many plant foods (spices and citric
acid) and in some animal foods (lysozyme), and the metabolites of food-grade
microorganisms (metabolites of starter-culture bacteria such as lactic acid bacteria and
some yeasts). Many of the organic compounds which have stirred interest are antimicrobial
metabolites of bacteria used to produce, or associated with fermented foods.
In fermentation, the raw materials are converted by microorganisms (bacteria, yeasts
and molds) to products that have acceptable qualities of food. In a natural fermentation,
the conditions are set so that the desirable microorganisms grow preferentially and
produce metabolic byproducts which give the unique characteristics of the product. When
the yield is unstable and where the desired microorganism might not grow, or where
pathogenic microorganisms might also grow, a controlled fermentation is used. In a
controlled fermentation the fermentative microorganisms are isolated and characterized,
then maintained for use (starter culture). Starter cultures are added to the raw materials
in large numbers and incubated under optimal conditions. In common controlled-fermented
products such as sauerkraut and yogurt, lactic acid is produced by the starter culture
bacteria to prevent the growth of undesirable microorganisms in the non-sterile, raw
materials, and helps to make the products shelf-stable [2].
The consumption of fermented foods has increased greatly since the 1970's. This includes
common foods like yogurt, buttermilk and fermented sausages as well as ethnic foods such
as kefir, kumiss and tofu. One of the reasons for the increase in the consumption of
fermented foods is because consumers consider these foods to be healthy and natural. The
consumption of live cells of desirable microorganisms in the billions and their metabolic
products in fermented foods does not cause any panic or distress in the safety-concerned
consumers. These foods have been around for thousands of years, and therefore have
withstood the test of time[3]. A natural next step would be to incorporate the
same antimicrobial compounds naturally found in fermented foods into other foods in lieu
of "chemical" preservatives.
In the last decade, there has been extensive research into the use of lactic acid
bacteria to control pathogenic and perishing microorganisms in food. There are three main
conditions for an optimal lactic acid fermentation: Addition of a sufficient amount of
fermentable carbohydrates; Reduced pO2 during the fermentation process and storage of the
fermented product, and; Rapid multiplication of the starter culture and sufficient
production of lactic acid. In addition, different factors are known to influence the
preservative action of lactic acid bacteria in meat products. The main factors for
successful preservation of meat products are: A low pH (<4.5 to prevent growth of unwanted bacteria); A substantial amount of non-dissociated organic acid molecules; Buffering capacity of the substrate; Hydrogen peroxide; Competition with other bacteria for nutrients; Production of antibiotics and bacteriocins, and; Decreased redox potential[4]. A major concern for
refrigerated foods is the possibility of temperature abuse during distribution, retailing,
or by the consumer.
The simplest application of lactic acid bacteria involves adding lactate salts such as
sodium lactate and potassium lactate to foods. These salts can inhibit the growth of
psychotropic pathogens and have been shown to protect refrigerated poultry and seafood
against Clostridium botulinum, Listeria monocytogens, and Aeromonas
hydrophila. The increased shelf life is further complemented by protecting against the
adverse effects of temperature abuse[5].
The bacteriocins produced by lactic acid bacteria have gained much attention as
potentially useful food additives against food-borne pathogens. Class I bacteriocins
(lantibiotics) undergo extensive post-translational modifications and contain very unusual
amino acids. Nisin, for instance, is a 34-residue peptide that is very active against most
Gram-positive bacteria. In contrast, class II nonlanthionine-containing bacteriocins such
as the lactococcins, the pediocins, the lactacins and leucocin A, are 36-44 amino acid
peptides that are minimally modified. Most class II bacteriocins are potent against Listeria
monocytogens[6] .
Nisin was the first bacteriocin derived from fermentation of a lactic-acid bacterium and
was approved by the FDA in April 1989 to prevent the growth of botulism spores in
pasteurized process-cheese spreads. Nisin does not inhibit Gram-negative organisms, yeasts
or fungi, but does inhibit most Gram-positive organisms including spore-formers such as Clostridia
botulinum and heat-resistant spoilage organisms. Nisin is used by Alta in their all
natural preservative line of foods. Nisin offers processors a "clean" label as
well as extending refrigerated shelf life by 14 to 30days depending on the product[5].
While most bacteriocins are produced only during exponential growth, nisin is produced in
large amounts after cells reach their stationary phase [7], making it
appropriate for foods in which lactic acid bacteria are not expected to grow after
processing.
Piscicolin 126 is a bacteriocin that has been proven to be stable at high temperatures at
acidic pH values. While nisin has had mixed success with meat preservation, varying with
meat product and target organism, piscicolin 126 has shown more promise. Inclusion of
piscicolin 126 in a commercial ham paste inhibited the growth of Listeria monocytogens
for up to 14 days of storage at the abuse temperature of 10ụC. While nisin failed the
same test; ATLA 2341, a shelf life extender with antilisteral activity, exhibited less
inhibitor action against Listeria monocytogensthan piscicolin 126[8].
In a recent paper, Coventry et al[9] were able to extract nisin, pediocin
PO2,brevicin 286 and piscicolin 126 from fermentation media. While they only showed
partial success, their procedure shows merit. The extraction of bacteriocins is important
for the enhancement of preservation in non-fermented foods as well as foods in which
inoculation with lactic acid bacteria is not appropriate.
Silage making is an important method of crop-preservation. Commonly ensiled crops include
grasses, corn, and legumes such as alfalfa. Silage fermentation depends on lactic acid
bacteria present on the crop, fermenting water-soluble carbohydrates to lactic and acetic
acids. The resulting low pH and toxicity of undissociated acids restricts the growth of
unwanted microbes and ensures good preservation. A common problem when ensiling alfalfa,
however, is that water-soluble carbohydrate levels are insufficient to allow adequate
lactic acid production. Alfalfa contains significant quantities of starch which could be
used by an amylotic inoculant strain of Lactobacillus plantarum, a common silage
bacteria. Fitzsimons et al[10] recently presented a paper describing a
recombinant alpha-amylotic Lactobacillus plantarum strain which has considerable
potential for silage of crops such as alfalfa in which starch is present with low levels
of water-soluble carbohydrates.
Many applications of lactic acid bacteria for food preservation exist. Several of these
are already in common use, and others are being researched and tested for efficacy and
safety.
Whereas many antibiotics disable or kill pathogens over a series of days by inhibiting
essential enzymes, most gene-encoded antimicrobial peptides (bacteriocins) kill
microorganisms rapidly by destroying or permeating the microbial membrane and impairing
the ability to carry out anaerobic processes. These peptides are therefore unlikely to
face the same antimicrobial resistance mechanisms that limit current antibiotic use[6].
As with any other biotechnological development, there is a concern over the safety of
purposefully inoculating food with genetically modified bacteria. Klijn et al determined
that based on the low survival in the environment and the regular gene transfer rates,
consumption of lactococci that are genetically modified by deletion of genetic information
or another self-cloning procedure in a fermented milk product does not influence the
potential hazard to the consumer.
In contrast, Hertel et al recently reported the results of their trial of transformed Lactobacillus
curvatusLTH1432. While no plasmid transfer could be induced even under optimal
laboratory conditions, the conditions in the meat matrix permitted an efficient transfer.
This result suggests that a case by case safety assessment is necessary to test the safety
of food grade organisms.
Because lactic acid bacteria and their byproducts have been present in food since the
early days of civilization, consumers readily accept them. Advanced molecular techniques
which allow the development of new strains with new features bring with them issues of
safety. Because of their mode of action, bacteriocins are poor candidates for resistance
development. The stability of transformants, however, is not so certain, and more research
is needed to prevent transfer of transformed genes.
Lactic acid bacteria and their byproducts are currently present in many of the foods we
consume. For this reason, they are regarded as safe and natural by consumers. Metabolic
byproducts of lactic acid bacteria have been shown to inhibit the growth of several
important pathogens, and increase shelf life beyond current "chemical"
preservatives.
Because lactic acid bacteria already preserve foods such as cheese and milk, it makes
sense to try and inoculate them into other foods as well. For foods in which lactic acid
bacteria inoculation is not suitable, protocols for efficient extraction of antimicrobial
compounds are being investigated. Purification of the antimicrobial compounds provides a
further protection against unwanted transfer of transformed genes to other strains of
bacteria.
Simple modifications such as deletion of genetic information or self-cloning have shown to
be safe in environmental tests, despite high rates of gene transfer among lactic acid
bacteria. More complex techniques such as transformation with new genes requires
case-by-case tests to determine the possibility of unwanted post-treatment gene transfer.
Lactic acid bacteria have been shown to be safe and effective. Research is ongoing to use
new strains in new domains. Besides being less potentially toxic or carcinogenic than
current antimicrobial agents, lactic acid bacteria and their byproducts have ben shown to
be more effective and flexible in several applications.
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