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Preservatives in Food Products – Review

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International Journal of Pharmaceutical & Biological Archives 2011; 2(2): 583-599

REVIEW ARTICLE

Preservatives in Food Products – Review

K.Seetaramaiah, A.Anton Smith*, R.Murali, R.Manavalan

Department of Pharmacy, Annamalai University, Annamalai Nagar – 608002, Tamil Nadu, India.

Received 22 Jan 2011; Revised 17 Feb 2011; Accepted 13 Mar 2011

ABSTRACT

The objective of this review is to examine the available safety/toxicity literature on preservatives like
olive polyphenols, microbial fermented tea, essential oil, parabens, and the micro organisms.
Antimicrobial activities of microbial fermented tea are much less known than its health beneficial
properties. These antimicrobial activities are generated by fermentation process with tea leaves as
substrates. The essential oil of Thymus vulgaris L. showed highest antifungal efficacy, significant anti-
aflatoxigenic efficacy, fungi toxicant and synthetic fungicidal effect. Parabens are readily absorbed
through the gastrointestinal tract and are rapidly excreted in the urine. The Enterococcus genus, a member
of the lactic acid bacteria (LAB) is found in the intestines of humans and other animals. Although
sometimes associated with pathogenicity, these bacteria are used as probiotic cultures and nowadays
extensively used for the production of bacteriocins the enterocins. This study gives a proof of using
preservatives and its efficacy in food and food products.
Key Words: Toxins, Food, Safety, Preservative

INTRODUCTION: of food, preserve the food characteristics like

odor, taste and food is preserved for a long time.
Food is an essential thing for human survival.
Except our own garden plants, all the food used The olive is the fruit of an evergreen olive tree
today has some preservatives. Preservatives are that grows in the temperate climate of the
the substances, which are used to prevent food Mediterranean region [1]. The content of phenolic
spoilage from microorganism. Food preservation compounds in olives and olive oil depends on the
is used from the ancient times. This will inhibit cultivars and the ripeness of the fruit at the time of
the growth of microorganisms like bacteria and harvest. The oil contained in olives is normally
fungi. extracted by a multi-stage process, which involves
crushing the whole olive (including the pits),
Food preservatives becomes an essential thing kneading the resulting paste, collection of the free
nowadays, this plays an important role during flow oil, pressure separation and collection of
food transportation. This will preserve the food residual oil in the grinds, and separation of solids
for a long duration from the spoilage. Each and and vegetation water from the olive oil. Generally
every packaged food items has some virgin olive oil has a free acidity, expressed as
preservatives, without them the food has no longer oleic acid, of not more than 2% as well as the
survive. Radioactive materials like cobalt-30 are other characteristics that correspond to those fixed
used as food preservative. Modern packaging for this category in this standard. Because of the
techniques includes vacuum and hypobaric strong antioxidant properties of the olive phenols,
packaging also acts as preservation technique. several investigators have attempted to isolate
Food preservatives aim to preserve the appearance phenols from the vegetation water, but because
the entire olive is macerated, the vegetation water
*Corresponding Author: A.Anton Smith, Email: auantonsmith@yahoo.co.in
 Anton Smith et al. / Preservatives in Food Products – Review

contains some undesirable substances from the chain length of the ester group increases, but since
crushed pits. solubility decreases with increasing chain length,
the lower esters (methyl and propyl) are the
Recently, several microbial fermented teas got practical choices for use in foods. Methyl and
noticed in the Western world, probably not only propyl parabens has been used as an antimicrobial
because of trade expansions between china and preservative in foods, drugs and cosmetics for
the west, but also because of several health over 50 years. There have been several previous
beneficial claims associated with microbial safety assessments undertaken on this substance
fermented tea. Recently, some higher plant by several agencies, including FAO/WHO, FDA
products viz. azadirachtin from Azadirachta and FEMA.
indica [2], carvone from Carum carvi [3] and
allylisothiocyanate from mustard [4] have attracted Lactic Acid Bacteria (LAB) is a diverse group of
the attention of microbiologists to search for some beneficial bacteria which have been inadvertently
newer phyto chemicals for their exploitation as used by mankind for thousands of years. They
antimicrobials. Such plant products would be have been used as starter cultures for preparation
biodegradable and safe to human health [5]. A few of an array of fermented dairy products which
studies reveal that extracts from microbial include yoghurt, cheese, buttermilk, kefir and
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

fermented teas contain natural antimicrobial many products indigenous to various regions of
components that have inhibitive effect on several the world. The preparation of these products has
food-borne pathogen and spoilage bacteria [6]. been documented in archaic texts of various
Application of natural antibacterial agents has regions such as ancient Iraq [7]. The LAB has
been increasingly noticed as a novel trend in been divided into many genera, and those
biological preservation of foods in recent years. important in food include Lactobacillus,
Essential oils (EOs) of some angiosperms have Lactococcus, Enterococcus, Pediococcus,
been investigated for their fungitoxicity against Leuconostoc and Streptococcus. Among them the
the toxigenic strain of A. flavus (Navjot 4NSt), a genus Enterococcus is of particular interest.
potent post harvest storage fungus of deteriorating
cereals and pulses.
The members of the genus Enterococcus are
Parabens, alkyl esters of PABA, are a class of found in many food products. They are most
antimicrobial agents used singly or in combination frequently present in many traditional European
to exert the intended antimicrobial affects against cheeses prepared mostly from raw ewes' or goats'
molds and yeasts. These substances can have milk, where they are thought to have initially
multiple biological effects, but it is generally made their way as contaminants from the feces of
considered that their inhibitory effects on animals, water or milking equipment and storage
membrane transport and mitochondrial function tanks, and thereafter became an important
processes are key for their actions. The parabens component of artisanal cultures [8]. These bacteria
meet several of the criteria of an ideal now play a fundamental role in the ripening of
preservative, in that they have a broad spectrum of these cheeses, probably by proteolysis, lipolysis
antimicrobial activity, are safe to use (i.e. and citrate breakdown, hence adding a unique
relatively non-irritating, non-sensitizing, and of taste and flavour to these products [9]. In addition
low toxicity), are stable over the pH range, and are to providing the peculiar taste and flavour, the
sufficiently soluble in water to produce the enterococci also act as protective agents against
effective concentration in aqueous phase. various pathogens, such as Listeria
Antimicrobial activity of paraben increases as the monocytogenes, a common pathogen found in
meat and dairy products.

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Table 1: Utilization of enterococcal strains for in-situ bacteriocin production
Typical target
Producing strain Enterocin produced Product tested Reference
organisms
Taleggio (Italian soft (Giraffa and
E. faecum 7C5 Undefined bacteriocin L. monocytogenes
smear cheese) Carminati, 1997)
(Giraffe et al.,
E. faecium 7C5 Undefined bacteriocin Milk Listeria innovua
1995)
Lactococcus lactis MG 1614 Enterocin A Cottage cheese L. monocytogenes (Liu et al., 2008)
(lzquierdo et al.,
E. faecium WHE 81 Enterocins A and B Munster cheese L. monocytogenes
2009)
Goat’s milk and Jben
Enterocins L50A and (Achemchem et
E. faecum F58 (Moroccan goat milk’s L. monocytogenes
B al., 2006)
cheese)

E. faecalis A-48-32 Enterocin AS – 48 Non-fat hard cheese Bacillus cereus (Munoz et al.,
2004)
E. faecalis A-48-32 Skimmed milk and
Staphylococcus (Munoz et al.,
Enterocin AS – 48 Non-fat unripened soft
aureus 2007)
cheese
E. faecium CCM 4231, E. Ebterocin CCM 4231,
Spanish style dry (Callewaert et al.,
Faecium RZS C13 and enterocin 13 and Listeria spp.
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

fermented sausages 2000)

Lactobacillus sakei CTC494 sakacin K
Enterococcus casseliflavus IM Cacciatore (Italian (Sabia et al.,
Enterocin 416K1 L. monocytogenes
416KI sausages) 2003)
Dry fermented (aymerich et al.,
E. faecium CTC492 Enterocins A and B L. innocua
sausages 2000a)
E. faecium CTC492 and L. Enterocins A and B (Aymerich et al.,
Cooked pork L.sakei CTC746
sakei CTC 494 and Sakacin K 20020

2. TYPES OF FOOD PRESERVATIVES 2.3. Microbial preservatives:

Food preservatives may be classified as Natural, Microbial preservatives are the preservatives
Artificial, and Microbial preservatives. which inhibit the growth of bacteria and fungi, or
2.1. Natural Preservatives: anti-oxidants such as oxygen absorbers, which
Natural food preservatives are good to our health. inhibit the oxidation of food constituents.
They do not harm our health. Natural
preservatives are sugar, salts, vinegar and 3.DESCRIPTION, SPECIFICATIONS,
rosemary extracts. Well known preservative OCCURRENCE AND SOURCE
techniques are refrigerating, boiling, pickling, and 3.1. Natural preservatives:
dehydrating are used in kitchens An aqueous olive pulp extract, is a standardized
2.2. Artificial Preservatives: freeze-dried powder prepared as a byproduct
Artificial preservatives are the chemical substance during the processing of the pulp of olives (Olea
that stops the growth and activities of the europaea L.) for oil extraction. The powder has an
microorganisms and helps to preserve the foods odor of processed olives and a characteristic
for a longer time without affecting its natural aromatic sour/olive flavor. The powder is
characteristics. It includes Antimicrobial agents composed of 98–99% dry solids, including 1–2%
and Antioxidants. Antimicrobial agents are used citric acid and 6% poly phenols. Other
to prevent the action of micro organisms. Certain constituents of the extract include protein, fat and
antimicrobial agents are benzoates, nitrites, carbohydrates. The biologically important
calcium propionate, sorbates, EDTA and sodium constituents of olive pulp extract are polyphenols.
benzoates. Antioxidants are the agents which are Among the phenolics, the major constituent of the
used to prevent the oxidation caused in the food pulp extract is hydroxytyrosol (50–70%), while
material. Certain antioxidants are BHT, BHA, other polyphenols present include oleuropein (5–
formaldehyde and ethanol. 10%), tyrosol (0.3%), oleuropein aglycone and
gallicacid.

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3.1.1. Phenols in olives: 3.1.2. Tea extract:
Olive fruit is known to contain simple, as well as Both Puer tea and Fuzhuan brick-tea are microbial
complex phenolic substances. These phenolics are fermented black teas, where as Kombucha is a
responsible for the stability of the oil from fermented drink of tea extract supplemented with
oxidation and for the organoleptic properties [10]. sucrose and fermented with yeasts and acetic-acid
In olive oil, phenols are present at levels up to 1% bacteria. All three tea(s) have shown obvious
by weight, both as simple and complex antibacterial effects.
compounds. Hydroxytyrosol and tyrosol, as well
as the lipid soluble oleuropein and ligstroside Puer tea has not only a unique flavour but also
aglycones, are partially released (5–10% of the several health beneficial properties, such as
total in olives) from olives into the oil during suppressing fatty acid synthase expression [14],
production (crushing), while the substantial acting as an inhibitor of lipid and non lipid
proportion remains in the water phase (vegetation oxidative damage and also exhibiting metal-
water). binding ability, reducing power, and scavenging
effect [15]. Recently, anti mutagenic and
Vegetation water is a good source of phenolic antimicrobial activities of Puer tea were also
antioxidants (1–1.8% w/v), as about 90% of the reported [16]. Fuzhuan brick-tea is another kind of
phenols in olives are transferred to the water microbial fermented tea uniquely found in
phase during the pressing of the drupes. China [17].
Approximately 10–20% of the total phenol
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

content can be recovered; the only bioactive Kombucha is a slightly sweet-sour flavoured tea
catechol recovered is hydroxytyrosol [11]. In beverage, obtained by fermentation of sweetened
another study, Fernandez-Bolanos et al., [12] boiled tea with a mixed culture of yeasts and
reported extraction of 3 kg of hydroxytyrosol (90– acetic-acid bacteria [18]. Kombucha is also
95% purity) from 1000 kg of olives during liquid– frequently called ‘‘tea fungus’’ in the literature,
solid waste of two-phase (conventional) olive oil although there is actually no fungus involved in
processing. Recently, CreAgri Inc., has been the fermentation [19]. This beverage reportedly has
granted two patents for the recovery of a number of health benefits, against metabolic
hydroxytyrosol from olive mill water (US Patent disease, arthritis, psoriasis, constipation,
numbers 6,416,808 and 6,197,308). indigestion, and hypertension, but there are few
3.1.1.1. Hydroxytyrosol: solid scientific evidences available yet for its
Hydroxytyrosolol, also known as 3,4- efficacy [20].
dihydroxytyrosol or 3,4-dihydroxyphenylethanol, 3.2. Artificial preservatives:
is the major component of the phenolic fraction of Methyl paraben is a colorless crystalline or white
olive extract and olive oil; the presence of powder. It is odorless or has a faint characteristic
hydroxytyrosol has also been identified and odor and a slight burning taste. Methyl paraben is
quantified in wines [13]. Hydroxytyrosol is present resistant to hydrolysis in hot and cold water but
in olive oil either as simple phenol or esterified hydrolyzes in alkaline solutions. It is stable in air.
with elenolic acid to form oleuropein aglycone. Aqueous solutions of methyl paraben buffered at
Pure hydroxytyrosol is a clear, colorless, tasteless pH 3 and 6 showed no decomposition when
liquid and can be hydro soluble or lipo soluble. It heated for 2 h at 100 °C or for 30min at 120°C.
is considered as the most potent (as measured by Methyl paraben is an ester of p-hydroxy benzoic
ORAC) phenolic antioxidant of olive oil. acid. It is produced by the methanol esterification
3.1.1.2. Oleuropein: of p-hydroxybenzoic acid in the presence of
Oleuropein is a phenolic secoiridoid glycoside sulfuric acid. Aqueous solutions of methyl
found in the bark, leaves and fruit of the olive paraben, at pH 36, may be sterilized by
tree, as well as in some other genera of the autoclaving at 120°C for 20 min, without
Oleaceae. The most abundant phenolic substance decomposition [21]. Aqueous solutions at pH 3–6
in the drupe is a bitter glycoside that constitutes are stable (less than 10%decomposition) for up to
up to 14% of the fruit’s dry weight. about 4 years at room temperature. Aqueous
3.1.1.3. Tyrosol: solutions at pH 8 or above are subject to rapid
Tyrosol , a minor component of olive oil has a hydrolysis (10% or more after about 60 days of
faint sweet fruity-floral odor and a sweet but very storage at room temperature). Natural occurrence
weak taste. of methyl paraben has been reported in

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cloudberry, yellow passion fruit juice, white wine, Propyl paraben is an ester of PABA and is
botrytised wine and Bourbon vanilla. The amount produced by esterifying PABA with n-propanol,
of methyl paraben detected in cloudberry is using an acid catalyst such as sulfuric acid and an
around 0.15 ppm [22]. excess of propanol. It is stable in air and is
resistant to hydrolysis in hot and cold water, as
Propyl paraben is a stable colorless crystalline or well as in acidic solutions. Above pH 7,
white powder with practically no odor or taste. appreciable hydrolysis occurs.

Table 2: FDA approved uses of methyl and propyl parabens in food (21 CFR, 2000)

Permitted
Source Citation number Food category Use limits
functionality
Not to
21 CFR 150.141 Food Standards. Artificially sweetened Artificially
FDA Preservative exceed
fruit jelly, Optional ingredient sweetened
0,1%
FDA
Artificially
21 CFR 150.161 Food Standards. Artificially sweetened Not to
sweetened fruit Preservative
fruit jams. Optional ingredient exceed
preserves and jams
0.1%
21 CFR 172.515 Food additives. Permitted for direct
(12) Flavoring
addition to agents and food for human consumption.
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

FDA No restriction agents and CGMPa

Subpart F-Flavoring agents and related substances.
adjuvants
Synthetic flavoring substances and adjuvants
21CFR 181.23 Prior sanctioned ingredients. Certain
FDA substances employed in the manufacture of food – No restriction (12) Antimicrobial CGMPa
packaging materials. Antimycotics
(2) Antimicrobial CGMPb
FDA 21 CFR 184.1490 Direct food substances affirmed GRAS. No restriction
agent 0.1%
FEMA FEMA GRAS No. 2710 No restriction Preservative 0.1%

3.3. Microbial preservatives: of preserving foods. Food preservatives can also

With the discovery of bacteriocins, the use of enhance the appearance of food items as well as
enterococci as starter cultures or co-cultures has add nutritional value.
been studied by various researchers not only for 4.1. Economical:
their organoleptic properties, but also for their There is no such thing as a broad spectrum natural
negative effect on food pathogens by production preservative. However, there are natural items that
of enterocins [23], as shown in the table 1. If the can have preservative qualities. These can reduce
bacteriocin producing enterococcal strain has the microbial activity (such as essential oils).
properties that make it suitable for use as starter The olive fruit, its oil and the leaves of the olive
culture such as high acid and flavour production, tree have a myriad of medicinal and other uses.
then it offers a double advantage of being used as Primarily, olives are used for their oil or as table
sole culture for both fermentation and food olives and are an important part of the
preservation However, if the enterocin producing Mediterranean diet. Because of their organoleptic
strain is being used along with another starter characteristics, olives require processing prior to
culture, then it should not interfere with the acid consumption [24]. In both fruit and oil, the
and flavour producing properties of the starter phenolics constitute a complex mixture, although
strain, otherwise the preservative effect of the there are some notable differences in composition
enterocin will be nullified by a low quality between the two that are attributed to a series of
product. chemical or enzymatic alterations of some phenols
during oil extraction. These modifications include
4. APPLICATIONS hydrolysis of glycosides by glucosidases [25],
Preservatives are natural, artificial and microbial oxidation of phenolic compounds by polyphenol
are used to maintain the shelf life and oxidases and, the polymerization of free
characteristics of various food items. Methods of phenols [26]. The quality of virgin oil is affected by
preserving foods have been used for centuries and the presence of phenolic compounds in olive
include natural techniques such as smoking fish fruits, as these compounds are partly responsible
and meat as well as adding salts. Refrigerating and for the stability and sensory characteristics.
freezing food items also falls under natural ways
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Parabens is widely used as an antimicrobial product formulations and maximum use
preservative in cosmetics, food products and concentrations for methyl, propyl parabens were
pharmaceutical formulations. It may be used 6606, 5868 and 25%, respectively [32]. Commonly,
either alone, in combination with other parabens, formulations contain parabens in concentrations
or with other antimicrobial agents. In cosmetics, up to 1%. Methyl paraben individually or with
methyl paraben is the most frequently used other parabens is used in all 13 product
antimicrobial preservative. formulation categories. Products containing these
4.2. Cosmetics: ingredients may contact the skin, hair and scalp,
The cosmetic products are basically the lips, mucosa (oral, ocular and vaginal), axillae and
derivatives of either suspensions or emulsions. nails. Products containing parabens may be used
Both the natural and microbial preservatives have on an occasional or daily basis and their use may
not ability to survive in the either suspensions or extend over a period of years. Frequency of
emulsions. Hence both the natural and microbial application and duration of exposure may be
preservatives are not preferred in the cosmetic continuous. Methyl paraben is the most used
products. Parabens are widely used cosmetic preservative in cosmetics [33]. In an investigation
preservatives present in a large variety of of 215 cosmetic products, a maximum of 0.32%
products, including face, body and hand creams, methyl paraben and 0.32% propyl paraben were
lotions and moisturizers; eye makeup products; present in paraben positive cosmetics. Of all
foundation and other makeup products; night paraben-containing cosmetics tested, 98%
creams and lotions; cleansing products; hair contained methyl paraben.
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

conditioners; bubble baths; shampoos; mud packs; 4.3. Food and food products:
underarm deodorants; skin lighteners; and Because of artificial microbial preservatives have
sachets [27]. Methyl and propyl parabens are the low toxicity and effective antimicrobial activity,
most commonly used preservatives in parabens, including methyl paraben, have been
cosmetics [28]. Parabens are found in all types of used in food for more than 50 years. Under FDA
formulations and have a use in over 13,200 regulation, methyl and propyl parabens are
formulations [29]. Concentrations of parabens are generally recognize as safe (GRAS) when used as
usually less than 0.3%, with the most common chemical preservatives in foods, with a use limit
preservative system containing 0.3% methyl of 0.1% (21 CFR 184.1490). Types of food that
paraben and 0.1% propyl paraben but may range may contain parabens include alcoholic
up to 1%. Parabens formulate well because they beverages, frozen dairy products gelatins, grain
have no perceptible odor or taste, are practically products, jams, jellies, marmalades, mincemeat,
neutral, do not produce discoloration, and do not olives, pickles, relishes, preserves, processed
cause hardening or ‘‘muddying’’ [30]. fruits and vegetables, tomato pulp, tomato puree,
catsup, fruit juices, soft drinks, puddings,
The popular use of paraben preservatives in seasonings, soft candy, sugar substitutes, syrups
cosmetics and toiletries arises from their low and sweet sauces [34]. Parabens are used in coffee
toxicity, broad spectrum of activity, worldwide extracts, fruit juices, pickles, sauces, soft drinks,
regulatory acceptance, biodegradability, and low processed vegetables, baked goods, seasonings,
cost. Other advantages of parabens are found in sugar substitutes and frozen dairy products at
their low tendency towards absorption in concentrations of between 450 and 2000 ppm [35].
commonly used plastics of primary packaging In a LSRO/FASEB [36] report it is stated that
material. methyl paraben is not reported to be used in fats
and oils, processed fruits, or alcoholic beverages.
Taking advantage of each paraben’s solubility Higuera-Ciapara and Nieblas [37] reported the use
characteristics, various concentrations of methyl of hydrogen peroxide- methyl paraben for
paraben and propyl paraben can be added to the preservation and stability of corn tortillas held at
base’s water and oil phases, respectively. These room temperature.
favorable characteristics make the combination of
methyl paraben and propyl paraben the most Methyl paraben is also permitted for use in certain
frequently used preservative system [31]. standardized foods including artificially
sweetened fruit jelly (21 CFR 150.141) and
According to the industry’s voluntary artificially sweetened fruit preserves and jams (21
submissions to the FDA in 1981, the number of CFR 150.161) at a level not to exceed 0.1% by

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weight of the finished food and are mentioned in 26.66%) in broccoli, tender onion, and broad
table 2. Methyl paraben, propyl paraben and butyl bean, cucumber, and red pepper juices.
paraben are permitted as direct food additives for
use in synthetic flavoring substances and The genus Bacillus and other related genera such
adjuvants in the minimum quantities required to as Paenibacillus and Alicyclobacillus consist of
produce their intended effects (21 CFR 172.515). heat resistant endospore forming bacteria. Though
As indirect food additives, methyl paraben and not pathogenic have often been involved in food
propyl paraben are permitted by prior sanction as spoilage and, therefore, need to be controlled.
antimycotics in food packaging materials with no Enterocin AS-48 has been found not only to
limit or restriction (21 CFR 181.23). successfully control the vegetative cells of
Bacillus spp. but also greatly increases the heat
In addition to the in-situ production, purified or sensitivity of endospores, thereby reducing the
semi purified forms of enterocins have also been time and temperature of heat treatment during
explored as preservatives in various foods, processing. This can also improve the quality of
especially in non fermented products. Enterocin the food products which may be affected due to
AS-48 appears to be the most promising candidate intense heat treatment.
in this regard as evident. It is the first enterocin
which was purified to homogeneity and The endospores of Alicyclobacillus spp. are
characterized, and is the most extensively studied thermo acidophilic and normal heat treatments
enterocins. Similarly enterocins A and B have applied during processing of fruit and vegetable
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

been tested as food preservatives especially in products are insufficient for their complete
meat products due to their strong anti-listerial destruction. They, therefore, grow during storage
activity. and are responsible for spoilage causing off-
flavours in the product [41]. The potential of
There is an increasing trend nowadays to consume enterocin AS-48 to control the vegetative cells
ready-to-eat fruit and vegetable products, with and spores of A. acidoterrestris DSMZ 2498
minimal processing and preservatives. However, strain was tested in freshly prepared or
the consumption of such products is not without commercial fruit juices. The enterocin was added
risk, especially the products which have high pH at a rate of 2.5 μg/ml to freshly made apple and
are more prone to contamination by pathogens. A orange juices.
number of disease outbreaks have been reported
due to the consumption of such products [38]. Use In addition to the spore forming bacteria, the
of enterocin AS-48, as a safe, natural efficacy of broad range enterocin AS-48 has been
antimicrobial offers a good alternative to investigated against many other Gram-positive
conventional preservatives to prevent the risk of and Gram-negative bacteria in different vegetable
food borne outbreaks [39]. and fruit products. L. monocytogenes has been an
organism of concern found as a contaminant in
A series of experiments by Grande et al. [40] many vegetable products, causing food poisoning.
investigated the stability of enterocin AS-48 in To explore the effect of enterocin AS-48 on this
fruit and vegetable juices. The results indicated pathogen, fresh alfalfa sprouts, soybean sprouts,
that a variable loss in antimicrobial activity and green asparagus were artificially inoculated
occurred when a concentrate of enterocin AS-48 with L. monocytogenes CECT 4032 before
was diluted 10 times in selected fruit and treatment with different concentrations (5, 12.5 or
vegetable juices. Among the vegetables, no loss in 25 μg/ml) of enterocin AS-48. The samples were
residual activity occurred in cabbage juice, stored at 6, 15 or 22 °C. The results revealed that
whereas some loss of activity occurred in celery at 6 and 15 °C, the pathogen remained below
juices (85.71% of control treatment). This was detection levels for 5 days at all concentrations of
followed by pumpkin, egg plant, lettuce, spinach, bacteriocin tested in contrast to the control where
leek, green bean, and avocado juices (from 40.05 the counts were always above detection limits. At
to 57.14% of control treatment), with endive and 22°C L. monocytogenes was not completely
asparagus juices showing the lowest activity. inhibited, however, the counts were still
However, doubling the bacteriocin concentration significantly lower (Pb: 0.05) as compared with
revealed some bacteriocin activity (9.16 to the control. The studies on the synergistic effect of
enterocin AS-48 along with other treatments have

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 Anton Smith et al. / Preservatives in Food Products – Review
been further extended to control Gram-negative
bacteria in vegetable products. This effect was monocytogenes and S. aureus which were
investigated in soybean sprouts treated with artificially inoculated (103 cfu/g) in model
selected species of enterobacteria and tested for sausages in independent trials. The enterocin was
synergism. When tested against S. enterica, no either added in a semi purified form or the
significant reduction (Pb: 0.05) was found in producer strains were inoculated for in-situ
samples treated with AS-48 alone. In contrast the enterocin production during preparation of model
combined treatment of heating at 65°C, 25μg/ml sausages. The results indicated that approximately
enterocin and alkaline pH (9.0), significantly (Pb: 40 μg/ml of crude bacteriocin preparation was
0.05) reduced the viable counts during storage at required for complete inhibition of the pathogens
15 °C for 48h. Similarly combined treatment of during storage period of 9 days. The addition of
enterocin AS-48 with of the selected preservatives producer strains (107 cfu/g) during preparation of
(lactic, polyphosphoric and peracetic acids, model sausages also helped in controlling the
sodium hypochlorite, hexadecyl pyridinium growth but complete inhibition could not be
chloride and hydrocinnamic acid) significantly achieved [46].
reduced (Pb: 0.05) the S. enterica counts during
storage at 15 °C for at least 48h. 4.4. Pharmaceutical applications:

Milk is considered as a complete food and an Parabens were first employed as preservatives in
excellent medium for the growth of various types pharmaceutical products in the mid-1920s [47].
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

of microorganisms as well. Therefore, during Parabens have been incorporated as preservatives

production and transport many microbes find their in a variety of drug formulations. Combinations of
way as contaminants into milk, and these parabens are more active than individual
microbes often include pathogens such as parabens [48]. Parabens are or have been used in
staphylococci and Listeria spp. These micro- suppositories, anesthetics, eyewashes, pills,
organisms may survive due to poor processing or syrups, weight gaining solutions, injectable
during preparation of such products as raw milk solutions, and contraceptives. Use concentration
cheese, and therefore can cause food poisoning varies from product to product but seldom exceeds
outbreaks [42]. The use of enterocins in dairy 1%. Methyl paraben is used in injections (0.065–
products can provide a natural hurdle in the 0.25%), ophthalmic preparations (0.015–0.05%),
growth of microorganisms. The enterocins can oral solutions and suspensions (0.015–0.2%),
either be produced in-situ by the addition of topical preparations (0.02–0.3%) and vaginal
enterocin producing cultures in the dairy products, preparations (0.1–0.18%) [49]. Golomb and
or by the addition of crude or purified Shipigelman [50] described the advantages of using
preparations. parabens, which make them suitable for imparting
antibacterial properties to implanted biomaterials.
In addition to dairy products, other products of
animal origin i.e., meat, poultry and sea foods, are
also highly prone to microbial growth because of Methyl and propyl parabens are used in a number
pH and nutrients. The microbes may range from of over-the-counter (OTC) drugs (21
spoilage organisms to food pathogens such as CFR310.545). The Ophthalmic Drug Panel of
E.coli, Salmonella spp., Campylobacter spp. and FDA’s Bureau of Drugs has determined that these
L. monocytogenes [43]. Due to the presence of such two ingredients, if used alone, are unsuitable as
organisms in meat products, many food borne preservatives in OTC ophthalmic products
outbreaks have occurred [44]. Therefore, the use of because they are irritating to eyes if used at
enterocins in the preservation of meat, poultry and concentrations effective against micro-organisms.
fish products has potential especially as nisin is Other OTC panels have concluded that methyl
not effective as a preservative in meat products paraben is a safe and effective preservative in
and it also has reduced activity against concentrations of 0.1–0.2% in products for
L.monocytogenes [45]. The use of broad-spectrum anorectal application and other antimicrobial
enterocins such as AS-48, or the strongly anti- uses [51].
listerial enterocins such as A and B have potential
in meat preservation. The effectiveness of
enterocin AS-48 has been tested against L.

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A range of different procedures are available for
5. DISCUSSION estimating the dietary intake of food
5.1. Estimation methods of food preservatives: preservatives [52]. The various methods are
summerised in (Table 3).
Table 3: Comparison of different methods for estimating intake
All dietary sources Reaction with food Analysis of samples Intake data on
Method Cost
of exposure covered matrix allowed for required individuals provided
Per-capita Low No No No No
Food diary Medium Possibly Yes No (literature data) yes
Food
Medium Possibly Yes No (literature data) Yes
frequency
Total diet High No Yes Yes No
Duplicate
High Yes Yes Yes Yes
diet
Biomarker High Yes Yes Yes Yes

5.1.1. Per-capita method: the average daily intake of the preservative

In this approach, information is obtained from the calculated. The technique has the advantage that
food preservative and food manufacturing the information produced is derived from real
industries on yearly production or usage of the food stuffs prepared in a manner reflecting
preservative. This value is then divided by the consumer practice.
number of people in the population. The main 5.1.5. Targeted surveys:
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

advantage of the per-capita technique is that it is a In this method, analytical surveys are conducted
very cost-effective means of obtaining estimates on those food items which are known to contain
of average intake arising from preservative usage. the preservative of interest. This information may
5.1.2. Food diary records then be combined with data on national food
In this technique, individuals keep a record of all consumption figures to provide an estimate of the
the food items eaten during a study period, which average intake of the preservative. Alternatively,
is typically up to 10 days. This food consumption the analytical data may be utilized in combination
data is then combined with the expected with more detailed information, available from
concentrations of the preservative in each recent comprehensive studies on dietary habits of
foodstuff and the resulting daily intake calculated. individual adults and children to provide an
A number of variations on this theme are possible. indication of actual intake.
The concentration of the preservative in each food 5.1.6. Duplicate diet studies:
item may be estimated on the basis of information In this approach duplicate portions of each
provided by the food manufacturer or from the component (or occasionally just particular items)
results of analytical surveys in the literature. of an individual’s diet are purchased and prepared
5.1.3. Dietary recall and food frequency as for consumption. The duplicate samples are
Dietary recall studies are retrospective and then analyzed to provide and has the particular
involve establishing preservative intakes on the advantage of reflecting actual intake but is
basis of individuals’ recollection of intake of necessarily very expensive.
specific food items. As with the food diary record 5.1.7. Biomarker-based methods:
approach, these data are then combined with The application of biomarkers to the intake
expected concentrations of the preservative to estimation of preservatives, and other food
estimate daily intake. This again is combined chemicals, is an area of considerable current
with analytical data on the concentration of the interest. The principle of the technique involves
preservative in the target food stuffs and dietary measuring the compound of interest, or one of its
intake calculated. metabolites, in a body fluid such as blood or 24 h
5.1.4. Total diet study: urine samples. Initially, developmental work is
In this approach the types and quantities of food required to establish the quantitative relationship
that make up the average British diet are between the daily dietary intake of the compound
calculated from the National Food Survey (Peattie and its biomarker, e.g. the amount of the
et al., 1983). The major items are prepared as if biomarker excreted in urine in the following 24 h.
for consumption and then amalgamated into 20 Once this is known the concentration in a 24 h
separate food groups, e.g. bread and cereals, fish, urine sample may be used to estimate the dietary
milk, etc. Each group is chemically analysed and intake of the compound in the preceding 24 h.
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 Anton Smith et al. / Preservatives in Food Products – Review
This approach may then be employed in Chou, Lin, and Chung reported that conventional
surveillance studies by collecting 24 h urine fermentative processed teas have decreased
samples from the target population, measuring the antimicrobial activity with the fermentation time
biomarker concentration in each individual’s and this implies that the originally present tea
sample and thence calculating dietary intake. catechins or polyphenols lose their antimicrobial
Biomarker-based methods have been widely used activity during the enzymatic oxidation. In their
to assess occupational exposure to potentially study, B. subtilis, E. coli, Proteus vulgaris,
harmful chemicals in the work place [53]. Pseudomonas fluorescens, Salmonella spp. and S.
5.2. Analytical methods: aureus were used to test the antimicrobial activity
Many analytical methods have been reported for of various tea extracts. Among the six test
the determination of preservatives, including microorganisms, P. fluorescens was the most
[54]
spectrophotometric methods , gas sensitive to the extracts, whereas B. subtilis was
[55] [56]
chromatography , HPLC , ion the least sensitive. Extract of green tea, the
chromatography [57], HPTLC-UV, Fourier unfermented or non-oxidized tea, showed the
Transform Near-Infrared (FT-NIR), TLC and strongest antimicrobial activity followed by the
micellar electro kinetic capillary chromatography. partially fermented tea products such as black tea,
The chemometric methods are an effective way to the completely fermented (oxidized) tea, showed
analyze simultaneously several analytes [58]. the least antimicrobial activity. It was also noted
that extracts of oolong tea prepared in summer
There is sufficient qualitative and quantitative exhibited the strongest antimicrobial activity,
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

scientific evidence to determine the safety-in-use, followed by those prepared in spring, winter and
i.e., the acceptable daily intake (ADI) for aqueous fall [59].
pulp extract. Ordinarily, ADIs are derived by
applying an uncertainty factor to the no effect Mo et al. [60] have done a microbiological analysis
level in an animal study. Based on the NOAEL of on samples from the indigenously fermented Puer
2000 mg/kg/day from a 13-week study in rats and tea. Microbial counting and identification revealed
also from a maternal and developmental toxicity that A. niger was the dominating microorganism
study, and applying uncertainty factors of 10 for during the fermentation. Antimicrobial activity of
interspecies differences and 10 for interspecies fermentation samples shows inhibitory effect on
differences, an ADI for ingestion of olive pulp several food-borne bacteria, including spore-
extract by humans can be determined. The overall forming bacteria Bacillus cereus, Bacillus subtilis,
uncertainty factor of 100 was judged appropriate, Clostridium perfringens and Clostridium
based on the considerations of animal studies and sporogenes.
the fact that constituents of the extract are
frequently consumed from food. Application of In a similar manner to studying Puer tea, we
the uncertainty factor of 100 to the NOAEL of analyzed the microbiological composition and
2000 mg/kg/day yields a safe intake for humans of tested the antimicrobial activity of extracts from
20 mg of olive pulp extract /kg per day or 1200 the indigenously fermented Fuzhuan brick-tea
mg/day (for an adult weighing 60 kg). (unpublished data). Microbial counting and
identification revealed that Aspergillus spp.,
In summary, based on a critical evaluation of the Penicillium spp. and Eurotium spp. were the main
available human, animal, analytical, and other microorganisms isolated from the samples during
scientific studies, and a history of exposure and fermentation and Eurotium spp. was the
use of components of aqueous olive pulp extract dominating fungus during the fermentation.
through table olives, olive products and olive oil, Antibacterial tests of extracts of fermented tea
the consumption of the extract is considered safe showed inhibitory effect on several food-borne
at levels up to 1200 mg/day. bacteria, including spore-forming bacteria B.
cereus, B. subtilis, C. perfringens and C.
The tea catechins or polyphones have played the sporogenes.
role of inhibition of microbial growth. This doubt
can be excluded by the fact that the antibacterial The antimicrobial activity of Kombucha was
activity increases with the fermentation time; tested against a number of pathogenic
namely, the longer the fermentation is, the more microorganisms. Staphylococcus aureus, Shigella
antibacterial activity the sample has. Nevertheless, sonnei, Escherichia coli, Aeromonas hydrophila,

592
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 Anton Smith et al. / Preservatives in Food Products – Review
Yersinia enterolitica, Pseudomonas aeruginosa, The fungitoxicity of isolated Essential Oils (EO)
Enterobacter cloacae, Staphylococcus epidermis, was tested against the toxigenic strain of A. flavus
Campylobacter jejuni, Salmonella enteritidis, following poisoned food technique [61] using
Salmonella typhimurium, B. cereus, Helicobacter potato dextrose agar (Himedia Ltd., Mumbai) as
pylori, and Listeria monocytogenes were found to nutrient medium are summerised in table 8.
be sensitive to Kombucha. According to the Requisite amounts of the oil dissolved separately
literature on Kombucha, acetic acid is considered in 0.5 ml of 5% tween-20 were pipette aseptically
to be responsible for the inhibitory effect towards to different pre-sterilised Petri plates (100 mm
a number of microbes tested. However, kombucha diameter) containing 9.5 ml of PDA medium so as
exerts antimicrobial activities against E. coli, S. to procure the requisite concentration of 1.0 and
sonnei, S. typhimurium, S. enteritidis, and C. 1.5 μl ml−1 . For control set req u si ite amou nt of
jejuni, even at neutral pH and after thermal sterilized distilled water in place of oil was added
denaturation of Kombucha. This finding suggests to the medium. A fungal disc (5 mm diameter) of
the presence of antimicrobial compounds other the toxigenic strain of A. flavus, cut from the
than acetic acid or large proteins in Kombucha. periphery of seven days old culture with the help
of a cork borer, was inoculated aseptically to the
Among the tested Bacillus bacteria, tea centre of the poured Petri plates of treatment and
polyphenols show antibacterial effects towards control sets. The plates were incubated at 27±2 °C
Bacillus stearothermophilus, which is a for seven days. Diameter of fungal colonies of
thermophilic spore-forming bacterium. The heat treatment and control sets was measured. The
IJPBA, Mar - Apr, 2011, Vol. 2, Issue, 2

resistance of B. stearothermophilus spores is percentage mycelial inhibitions were calculated by

reduced by the addition of tea polyphenols. the mean value of colony diameters by the
Clostridium thermoaceticum, an anaerobic spore- following formula [62] and were presented
forming bacterium, also exhibits reduced heat in(Ttable 4).
resistance of its spores in the presence of tea Percentage of mycelial inhibition =
polyphenols. Epigallocatechin gallate, a main
Where, dc = average diameter of fungal colony in
component of tea polyphenols, shows strong
control set
activity against both B. stearothermophilus and C.
dt = average diameter of fungal colony in
thermoaceticum.
treatment set
Table 4: Antifungal screening of some higher plant essential oils of different family against the toxigenic strain of A. flavus.
Plant name Family Antifungal activity
1.0µl ml-1 1.5µl ml-1
Aegle marmelos Rutaceae 70.53±2.56 83.36±2.11
Ageratum conyzoides Asteraceae 83.93±3.05 95.30±2.02
Artemisia vulgaris Asteraceae 25.33±1.61 30.73±1.44
Callistemon lanceolatus Myrtaceae 66.83±2.84 81.66±2.54
Citrus reticulate Rutaceae 22.90±2.02 28.63±2.22
Commiphora mukul Useraceae 60.40±2.34 73.43±2.60
Curcuma longa Zingiberaceae 81.63±2.86 91.53±1.69
Eucalyptus citriodora Myrtaceae 67.30±2.82 81.732.28
Eupatorium connabinumlamiaceae Asteraceae 62.00±3.43 81.86±2.33
Hyptis suaveolens Lamiaceae 91.63±2.39 100.00±0.00
Murraya koenigii Rutaceae 35.06±1.92 52.36±2.65
Thumus vulgaris Lamiaceae 10.00±0.00 100.00±0.00
Ocimum gratissimum Lamiaceae 90.43±1.62 95.30±1.80
Zingiber officinalis Zingiberaceae 87.63±2.07 93.70±3.40

To find out the minimum inhibitory concentration

(MIC) at which the thyme oil showed absolute incubated for seven days at 27± 2 °C. The nature
fungi toxicity, experiments were carried out by the of toxicity (fungi static/fungicidal) of the essential
usual poisoned food technique [63]. Different oil was determined following Kumar et al. [64].
concentrations of the oil viz. 0.1, 0.2, 0.3, 0.4, 0.5, The inhibited fungal discs of oil treated sets were
0.6, 0.7, 0.8, 0.9 and 1.0 μl ml−1 were pr epared re-inoculated on fresh medium after washing with
by dissolving separately its requisite amount in distilled water and revival of their growth was
0.5 ml 5% tween- 20 and then admixing with 9.5 observed.
ml of PDA. The inoculated Petri plates were
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 Anton Smith et al. / Preservatives in Food Products – Review
The spectrum of fungi toxicity of thyme oil was
evaluated against eight fungi viz., F. oxysporum, Methyl paraben and propyl paraben have been
C. herbarum, C. lunata, A. terreus, A. niger, A. affirmed by the FDA as GRAS for direct addition
fumigatus, A. alternata and B. theobromae at its to food (21 CFR 184.1490) in concentrations up to
MIC i.e. 0.7 μl ml
−1 by the usual poisoned food 0.1% and by prior sanction as indirect addition via
technique on PDA as nutrient medium. packaging materials (21 CFR 181.23). Both
compounds are used to inhibit molds and yeasts in
The safety limit of thyme EO was determined by various foods. They are active against Gram
recording LC50 value on mice following positive and a few Gram negative organisms. In
Srivastva and Saxena [65]. Mice (Mus musculus L.) addition to FDA, the Joint AO/WHO Expert
with an average weight and age (35 g, 3months) Committee on Food Additives (JECFA) and the
were selected as test animal for the mammalian Flavor and Extract Manufacturer’s Association
toxicity experiments. Requisite amount of thyme (FEMA) has also approved the use of methyl and
EO was mixed properly with tween-80 and propyl parabens in foods mentioned in (Table 5)
distilled water (2:1) to prepare different solutions with a possible average daily intake of 0.237mg.
containing desired dose of oil from 100–300μl. JECFA in 1974 recommended that the group ADI
0.5 ml of each solution of EO was orally for the methyl, ethyl and propyl esters of p-
administered separately through a syringe with hydroxybenzoic acid was 0–10 mg/kg body
catheter to each set containing 12mice. In control weight/day. Usual and maximum use levels of
sets equal volume of stock of tween-80 and methyl paraben as FEMA food flavor ingredients
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distilled water (2:1) was given to mice. After 72 h, are given in Table 5. The FEMA reported possible
the mortality of the animals was recorded and average daily intake (PADI) of methyl paraben is
LC50 was calculated in terms of per kg body 0.22 mg.
weight of mice.

Table 5: FEMA use levels for methyl and propyl paraben

Maximum use level
Usual use level (ppm)
Food category (ppm) Methyl Paraben
Methyl Paraben Propyl Paraben Propyl Paraben
Baked goods 0.99 0.97 0.99 0.97
Fats and oils - 0.32 - 1.00
Frozen airy 0.00 0.06 0.01 0.08
Fruit juice - 0.10 - 0.10
Processed vegetables 1.00 1.00 1.00 1.00
Soft candy 0.00 0.06 0.01 0.08
Gelatin and pudding 0.00 - 0.00 0.01
Non-alcoholic
- - 0.01 0.01
beverages
Alcoholic beverage 0.00 - - 0.02
Milk Products 0.50 - 0.00 -
Cheese 0.00 - 0.00 -
Meat Products 0.00 - 0.00 -
Sugar substitutes 0.00 0.25 0.50 0.025

Use of paraben, including methyl paraben, is the Philippines, Sweden and Switzerland, have
covered by an EU Directive, the ‘‘European also approved the use of parabens as antimicrobial
Parliament and Council Directive No. 95/2/EC of food additives. In Italy, methyl paraben may be
February 20, 1995 on food additives other than used as a direct food additive.
colors and sweeteners”. Preservation of cosmetic
products with methyl-, ethyl-, propyl-, butyl- and The parabens are effective in acid, neutral and
benzyl paraben, with a maximum concentration of slightly alkaline solutions. Beyond pH 8,
0.8% (w/w), calculated as p-hydroxybenzoic acid hydrolysis can occur and this reduces preservative
is permitted by the Danish and EEC efficiency [67]. Parabens are known for their
regulations [66]. Parabens may be present in some inhibitory action on microbial growth [68]. Spore
cosmetics labeled as ‘‘hypoallergenic’’. Several germination is much more susceptible to parabens
other countries, including Canada, Japan, Norway, than vegetative growth in fungi or bacteria [69].
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 Anton Smith et al. / Preservatives in Food Products – Review
Parabens, including methyl paraben, have been properties of methyl paraben against a number of
found effective in low concentrations against strains are summarized in the Final Report on the
fungi and bacteria. Parabens are more active Safety Assessment of Methyl, Ethyl, Propyl and
against fungi than bacteria and are more active Butyl Parabens. Microbiological activity of
against gram-positive bacteria than gram-negative methyl paraben against different microbes is
bacteria. The antifungal and antibacterial summerised in (Table 6).

Table 6: Inhibitory concentrations of parabens

Organisms Concentration
MIC Value a
E. coli 2000
S. aureus 2000
P. aeruginosa 4000
MIC value b
E. coli 800
P. aeruginosa 1000
MIC (giving 50% inhibition of growth and uptake process) Concn (mM)
E. coli ML308-225 5.5
P. aeruginosa ATCC 9027 3.6
B.sublitis ATC6633 4.3
Inhibitory concentrations (µg/ml)
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C. albicans 1000
S. cerevisiae 1000
Trichophyton spp. 160
Penicillium spp. 500
Aspergillus spp. 600
Lethal concentrations (µg/ml)
C. albicans 5000
P.Chrysogenum 5000
A. Niger 5000
MIC value (µg/ml)
A. oryzae 600
Trichoderma lignorum 250
ACTT 8678
Sarcena hutea 4000
Enterobacter cloacae ATCC 23355 1000
Proteus vulgaris ATCC 8427 2000
MIC valuesc (µg/ml)
E.coli DC0 1400
E. coli DC2 (envelop defective) 1000
P. aeruginosa 799 1800
P. aeruginosa 799/61 (envelop defective muatant) 1000

Enterocins A and B can effectively check the effectively controlled a listerial strain, L.
growth of L. monocytogenes when incorporated in monocytogenes NCTC 10888. The enterocin
biodegradable films (alginate, zein and polyvinyl coated film effectively decreased the listerial
alcohol). When incorporated at a concentration of counts during first 24h, and the trend was
2000 arbitrary units/cm2 (AU/cm2) with air maintained for two weeks at refrigerated storage
packaging they effectively checked the growth of and for longer times at higher temperatures. A
L. monocytogenes in cooked ham for 8 days. With different trend was observed when fresh soft white
vacuum packaging the effectiveness was further cheese was packed in film coated with enterocin
increased showing no increase from inoculated 416K1. The pathogen counts were 1 log unit
levels of L. monocytogenes during15 days of lower than the control for up to 28 days under
refrigerated storage [70]. Enterocin 416K1, refrigeration, but only about a week at 22°C, with
produced by E. casseliflavus IM 416K1 when the control treatment decreasing by 0.5 log units
coated to a LDPE (low density polyethylene) film after 28 days [71].

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6. SUMMARY: or semi purified preparations of bacteriocins as

food preservatives have legal implications.
Based on a critical evaluation of the available Despite being produced by LAB, any new
human, animal, analytical, and other scientific bacteriocin intended to be used as food
studies, and a history of exposure and use of preservative is considered as an additive and
components of aqueous olive pulp extract through needs prior approval by the regulatory authorities,
table olives, olive products and olive oil, the requiring detailed safety information supported by
consumption of the extract is considered safe at toxicological data, proof of efficacy in foods,
levels up to 1200 mg/day. With the trend of description of manufacturing process, as well as
increasing use of natural and biological the safe maximum levels.
preservatives in food products, natural
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