Note: Descriptions are shown in the official language in which they were submitted.
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P~OPIONATES AND MUTILATES OF
PROPIONIBACTERIA AFFECTING MICROBIAL GROWTH
BRIEF SUMMARY OF THE INVENT ION
The present invention relates to chemical
substances which affect microbial growth. More
specifically, it relates to substances which inhibit
spoilage microorganisms in food products and to
substances which stimulate the growth and reproduction
of desirable microorganisms such as microorganisms
useful in the manufacture of cultured or fermented foods.
The need for improved methods of food and feed
preservation is great; activities of bacteria molds and
yeasts render millions of pounds of food inedible
annually and the problem is especially acute in
countries with inadequate refrigeration In the United
Kingdom, it is estimated that 50 million loaves of bread
are lost each year due to mold contamination (Soiler,
"Factors affecting the use of mold inhibitors in bread
and cake," Microbial Inhibitors in Food, Stockholm 1964,
p. 211). Feed also may spoil, be rendered unpalatable
or even become toxic as a result of microbial activity
(Rod ricks and Levitt, "Toxigenic Fungi", in Compendium
of Methods for Microbiological Fermentation of Foods,
Am. Pub. Health Assign. Ed MEL. Speck, 1976).
Among these spoilage microorganisms, the yeasts
and molds are economically the most significant because
they are so versatile from nutritional and
growth-temperature standpoints and because their spores
are so ubiquitous. Minimizing their effect in food
presently depends on sepsis and sanitation, exclusion
of oxygen and use of chemical additives (Ayes, Mandate
and Sardine, Microbiology of Foods, W. H. Freeman and
Co., 1980, p. 140).
These procedures are only partially
successful. For example, Suriyarachchi and Fleet (Apply.
and Environ. Microbial. 42:574, 1981) found that 45% of
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128 retail yogurt samples had yeast counts of greater
than 1000 per gram; nine different genera were
represented. Thus, almost half the product on the
market has a limited shelf-life as compared to products
made under good manufacturing practices where no greater
than 1 yeast cell per gram should be present initially
(Davis, Dairy In. 35:139, 1970). Such a product should
have a shelf life of at least 30 days at 5C (Kruger, J.
Dairy Sat. 59:344, 1975). Temperature abuse is common
in retail channels (Body felt and Davidson, J. Milk and
Food Tuitional. 38:734, 1975) and even longer shelf-life
times are being sought as product manufacturing sites
are being localized in fewer but larger plants which
distribute products over immense distances. For
example, one of the well-known brands of yogurt
available throughout the United States is manufactured
in only two localities. Distribution time therefore can
represent a significant proportion of the shelf life of
the product, depending on conditions of manufacture and
temperature history.
For some foods, bacteria are more important
spoilage agents than yeasts or molds. Cottage cheese
and market milk products such as pasteurized whole milk,
skim milk, half and half, and whipping cream are
25 examples of such products. Psychotropic bacteria, able
to grow at refrigeration temperatures (35 to 50F),
rapidly spoil these products in the commercial
marketplace. Such spoilage is a continuing and serious
problem for the dairy industry. The only control
practice-s now available to the industry for
psychotropic bacterial spoilage are sanitation and the
prevention of temperature abuse. The nature of the
industry and methods of product manufacturing and
handling, however, make these practices inadequate to
35 prevent the spoilage problem.
Prop ionic acid (CH3CH2COOH - The Merck
Index, Thea Ed ! Merck and Co., Inc., 1976) is known as a
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mold inhibitor, especially for use in spillage ~Draughon
et at, J. Food Sat. 47:1018, 1982), bread and in certain
food wrappers such as those for cheese (Ayes et at.,
Microbiology of Foods, W. H. Freeman and Co. 1980, p.
140; Moon, Pro. Am. Sock Microbial., 1981, p. 29).
Wilfred and Anderson ("Preappoints Control
Microbial Growth in Fruits, Vegetables", Food
Industries, 17, pp. 622-624, 726, 728, 730, 732, 734,
June 1945) reported that sodium preappoint incorporated
in concentrations of 0.2~ into nutrient broth at pi 4.5
inhibited Pseudomonas fluoresces along with some other
bacteria. Concentrations of 0.5~ at pi 5.0 inhibited
two different molds and at pi 4.5 concentrations of 2.0%
were needed to inhibit SaccharomYces ellipsoids yeast5 and concentrations of 3.0% were needed to inhibit
yeast. Thus, low
concentrations were not effective against yeast. For
figs, a dip into 15% calcium preappoint or incorporation
of 0.5% calcium preappoint in a fig puree inhibited
growth of mold on the figs. Young berries were treated
by dipping into 5% sodium preappoint or 10 percent
sodium preappoint or sprayed with a 10 percent
solution. These treatments inhibited mold growth.
Apple slices were treated with a 0.5 percent calcium
preappoint solution and these "proved to be less
susceptible to damage by molds than were similar slices
not treated with preappoint. However, The fruit carried
a prop ionic odor and tended to become more noticeably
gray than did untreated slices".
Peas were treated with a 5 percent preappoint
solution at pi 6.3. In this study, controls were
reported to be faded in color, to have a very
disagreeable odor, and to be very slimy. Preappoint
treated lots showed slight color change, no sliminess
35 and little abnormal odor. However, such differences
were not found by Wilfred and Anderson in a separate
study wherein peas were held for 4.5 hours after
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treatment with calcium preappoint (presumably a 5
percent solution) and then scalded, frozen, and stored
for one month. At the end of the storage period,
prop ionic flavor was not detected in the treated peas;
but the differences between controls and treated peas
for flavor and skin texture were insignificant.
Further, Wilfred and Anderson state that
experiments involving dipping of lima beans in 5 percent
sodium preappoint solution definitely inhibited
bacterial growth for a number of hours, as in peas.
Such inhibition of bacteria for a few hours is
meaningless for food preservation. The authors also
state "preappoints should be used with due regard to its
limitations, such as the pi of the product, its
microbial flora, and the concentration of preappoint
likely to impact foreign flavor or odor to the food."
An authoritative text (Handbook of Food
Additives, end Ed., CRC Press, 1972, pp. 137-141)
references the work of Wilfred and Anderson done in 1945
and then those who wrote this section in the CRC
Handbook reviewed other appropriate literature on
prop ionic acid, sodium preappoint and calcium preappoint
and concluded that "preappoints are more active against
molds than sodium bonniest, but have essentially no
activity against yeasts. They have little action
against bacteria with notable exception of their ability
to inhibit the organisms which cause rope." It is also
reported that preappoints "are suitable for yeast raised
as well as other baked goods" and "because preappoints
inhibit molds and spares yeast" they are used in breads
(the Handbook of Food Additives, end Ed., CRC Press,
1972, pp. 137-141).
Microbial metabolizes, especially so-called
antibiotics, which inhibit the growth of microorganisms
35 are well-known. Indeed, a large segment of the
pharmaceutical industry is based on the sale of purified
antimicrobial which find uses in medicine and-to some
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extent also in the food industry.
A considerable body ox literature exists on the
propionibacteria which produce prop ionic acid. Their
growth and metabolism have been reviewed (Hutting and
Reinhold, J. Milk Food Tuitional., 35:295, 358 and 463,
1972) as well as their contribution to the flavor and
microbiology ox Swiss cheese (Langsrund and Reinhold, J.
Milk Food Tuitional. 36:487, 531 and 593, 1973; 37:26,
1974). Early literature (Skew and Sherman, J. Dairy
Sat. 6:303, 1923) reported that propionibacteria
produced acetic and prop ionic acids and the production
of other volatile, namely acetaldehyde,
propionaldehyde, ethanol, propanol and dim ethyl sulfide,
by these bacteria was noted by Canaan and Bills (J.
Dairy Sat. 51:797, 1968). Dustily production by
propionibacteria was reported by Lee et at. (Can. J.
Microbial. 16:1231, 1970). The Handbook of Food
Additives, end Ed., (CRC Press, 1972), pp. 137-141,
provides background information on prop ionic acid and
its salts, including uses physical and chemical
properties, antimicrobial activity, safety, regulatory
status, applications, handling, storage and assay. The
same type of information on acetic acid and acetates is
presented in this reference on pages 147-150.
Propionibacteria also produce acetate along
with preappoint and COY as end products of lactic acid
metabolism (Allen et at., J. Bacterial. 87:171, 1962).
Propionibacteria are known to also produce substantial
amounts of succinic acid as well as acetic acid (Wood
and Workman, "Mechanism of Glucose Dissimilation by the
Prop ionic Acid Bacteria," Become. J. 30:618-623, 1936;
Wood and Workman, "The Relationship of Bacterial
Utilization of COY to Succinic Acid Formation",
Become. J. 34:129-137 (1940); Leaver, Wood and
35 Stjerholm, "The Fermentation of Three Carbon Substrates
by C. Propionicum and Propionibacterium", J. Bacterial.
70:521-530, 1955.
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Acetic acid is the main constituent of vinegar
and has a definite characteristic odor and flavor. The
Handbook of Food Additives, end Ed., (CRC Press, 1972),
refers to work which shows that homology of prop ionic
acid (such as succinic) have tastes and odors which
would be noticeable in foods such as baked goods.
Propionibacteria are used in the production of Swiss
cheese and here only a small amount (only about 10~ of
the total inoculum which is, in turn, only about 1-2~ of
lo the milk used) produces distinctive flavor
characteristics. Therefore, it is not anticipated that
a nutrient growth medium containing propionibacteria
could be used as a liquid suspension, after condensing
or after drying as an additive to foods or feeds without
producing an undesirable change in flavor or odor.
Propionibacteria are reported to produce an
anti viral component (Ramanathan, Read and Cutting,
"Purification of Propionin, An Anti viral Agent from
Propionibacteria", Pro. Sock Exp. Blot. Med.
123:271-273, 1966; Ramanathan, Walynec and Cutting,
"Anti viral Principles of Propionibacteria", Isolation
and Activity of Propionics 8 and C", Pro. Sock Exp.
Blot. Med. 129:73-77, 1968~
Despite the many reported techniques in the art
of food preservation and a great deal of ongoing
research concerning preappoints and propionibacteria,
there was until now no simple, natural additive
substance that could effectively inhibit such difficult
spoilage microorganisms as yeasts, molds and,
particularly, slime-producing psychotropic bacteria.
It is now discovered, quite surprisingly, that
prop ionic acid, in concentrations so low that flavor and
aroma are not adversely affected, inhibits yeast growth
in certain foods. It is also discovered that a mature
propionibacterium growth medium can provide prolonged
inhibition of yeasts, some bacteria and mold without
providing an undesirable flavor of succinic acid or
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prop ionic acid or vinegar. The unexpected findings
disclosed are especially dramatic in light of some of
the low concentrations which provide microbial
inhibition as described in following examples.
An antimicrobial food additive can be obtained
by growing propionibacteria, e.g. Propionibacterium
Sherman, P. freudenreichii, P. pentosaceum, P.
thin, P. arabinosum, P. rub rum, P. lensenii, P.
peterssonii, and related species (as identified in
Mali et at., Jan. J. Microbial. 14:1185, 1968) in a
milk cheese whey or broth medium or other suitable
nutrient mixtures. The resulting growth liquid is then
added to food and feed products to inhibit yeasts, molds
and spoilage bacteria. To facilitate storage and
shipment, the growth liquid may be dried to form a
powder, or frozen before use as an antimicrobial food
additive. Such powdered or liquid natural metabolizes
of propionibacteria can be incorporated into various
foods and feeds to render them less susceptible to
spoilage by growth and/or enzymatic activity of yeasts,
molds and bacteria.
The growth medium for such Propionibacterium
species may be formulated with milk or whey containing
yeast extractives or broth media containing appropriate
growth nutrients The growth liquid, after development
of the propionibacteria up to 106 to 101 cells per
ml, may be heat treated (pasteurized) to kill the
inoculated and adventitious bacteria prior to use in
liquid, condensed, dried, or frozen form. It is added
in various concentrations preferred between 0.01 and
10% of total weight) to food or feed where it functions
to inhibit yeasts, molds or certain bacteria. This
inhibition enables the shelf life and storage times of
the food or feed to ox increased.
Furthermore, it is discovered that the growth
liquid, after development of the propionibacteria, can
be mechanically separated into fractions, one of which
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inhibits yeasts, molds, and bacteria, and another of
which is stipulatory to the growth of useful bacteria
such as Streptococcus lactic, Streptococcus creamers,
Lactobacillus bul~aricus, Streptococcus thermophilus,
S Lactobacillus acidoPhilus, Leuconostoc species,
Lactobacillus Planetarium, and Pedro coccus cerevisiae.
It is therefore a general object of the present
invention to extend the shelf life of food products
subject to microbial spoilage
Another object is to provide a substance which
can be added to a food product to inhibit the growth of
mold, yeast and some bacteria without harming the
flavor, aroma, or other characteristics of the food
product.
A specific object is to extend the shelf life
of dairy foods, cultured foods, high acidity foods, and
specifically cottage cheese, yogurt, kisses, fruit
juice, sausages, and other ground meat products.
An additional object is to provide a method
which uses naturally produced substances, such as
metabolizes of Propionibacterium species elaborated in a
suitable growth medium, in the preservation of food and
feed.
It is also an object to provide antimicrobial
metabolizes of propionibacteria in a dried or frozen
form for simplicity of storage and shipment.
Further an object of the present invention is
to provide such a method to inhibit microorganisms which
are potentially injurious to human health.
A still further object is to provide an
antimicrobial food additive substance comprising a
growth mixture containing propionibacteria and
metabolizes thereof, with the bacteria being viable or
made not viable depending upon whether it is desired to
35 produce additional amounts of metabolizes, including
COY, after the antimicrobial substance is added to the
food product.
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12~3 !39~
g
Still another object is to provide a growth
medium in which propionibacteria can grow and produce
metabolizes having properties that effect the growth of
other microbes.
A related object is to separate and separately
utilize fractions of the mixture of metabolizes of a
propionibacterium culture, e.g. to inhibit and to
stimulate microbial growth, respectively
A related object is to provide a growth
stimulant useful in a starter culture of commercial
fermentation bacteria.
These and other objects will become
increasingly apparent by reference to the following
description.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
Figs. 1-4 are graphs showing the effect of a
supernatent fraction of a growth mixture of P. Sherman
on the growth of gram negative psychotropic cottage
cheese spoilage bacteria in lactose broth versus the
amount of the supernatent fraction applied.
DETAILED DESCRIPTION
There are several aspects to the present
invention as set forth below. It has been found
possible to inhibit spoilage microorganisms and thereby
extend the shelf life of many food products without
adversely affecting flavor or aroma by adding
preappoints, a growth mixture containing a
propionibacterium culture with its metabolizes or a
fraction of such a growth mixture. Such substances,
which should be widely accepted as safe for human
consumption, are surprisingly excellent inhibitors of
spoilage microorganisms.
Examples of the present invention are set forth
hereinafter. It is intended that they be only
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illustrative. Propionibacterium strains are available
from the American Type Culture Collection (ATTICS). The
other cultures are widely available or can be obtained
from Oregon State University, Corvallis, Oregon, without
cost.
A. ACIDS AND SALTS
As mentioned above, certain acids, in
particular prop ionic acid, were thought to have an
inhibitory effect on molds, certain bacteria, and
fungus, but no meaningful effect on yeasts. Prop ionic
acid is felt to be so compatible with yeast that it was
recommended as an additive for bread dough wherein yeast
vitality is essential
It has now been discovered that prop ionic acid
inhibits yeast in certain food products and is an
effective preservative in foods such as yogurt, wherein
yeast contamination is a major difficulty. Furthermore,
the amount of prop ionic acid required for success is so
unexpectedly low, as compared to other acids and past
reports, that prop ionic acid can be present in such a
small amount that it has no adverse effect on flavor or
aroma of the food product.
1. Succinic Acid
a. Mold and Yeast
The success of prop ionic acid in inhibiting
yeast is particularly surprising in view of the fact
that other closely related acids and salts are
ineffective. The following examples show that a closely
related compound, succinic acid, could not be used to
inhibit the growth of yeast.
EXAMPLE 1
Potato dextrose ajar (3.9 gym per 100 ml of
water) was prepared, autoclave, and the pi was lowered
by adding 1.4 gym of tartaric acid. Then, 8 different
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concentrations of succinic acid (0, 0.5%, 0.75%, I 2%
, 3%, and 4%) were prepared in the potato dextrose ajar
and the pi of each was adjusted to 3.5 with hydrochloric
acid; two plates of each were poured and dried for 24
hours and then one plate of each concentration was
inoculated with 0.2 ml of yeast cells and the other
plate of each concentration of succinic acid in potato
dextrose ajar was inoculated with 0.2 ml of mold cells.
The yeast and mold inkwell were prepared by
transferring a loop of either yeast or mold found
growing on commercially available yogurt products into
sterile water and then vortexing before use.
The succinic acid did not show inhibition of
mold or yeast in the above experiments as all of the
plates, including both the control plates with no
succinic acid and those plates with succinic acid,
supported growth of the mold and the yeast.
b. Bacteria
Despite the lack of success with yeast and
mold, it was found that succinate ions, from an addition
of succinic acid or soluble succinate salts, are
effective to inhibit Pseudomonas bacteria in certain
substances.
EXAMPLE 2
A second set of similar plates of varying
succinic acid concentrations in potato dextrose ajar
were prepared, as in Example 1. Differences were that
the second set of plates were adjusted to a pi between
4.5 and 5.5 USinCJ hydrochloric acid or sodium hydroxide
and the plates were inoculated with 0.2 ml of
Pseudomonas fluoresces.
The Pseudomonas control plates with no succinic
35 acid supported substantial growth after 30 hours and
growth was also observed on the 0.25~, 0.75%, and 1%
succinic acid plates. There was no growth observed on
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the 0.5~, 2%, 3%, or 4% succinic acid plates after 48
hours. Since the pi was adjusted to between 4.5 and
5.5, it is possible that inhibition was due to a low pi
of the plates, and not to the succinic acid, since
Pseudomonas does not grow well below pi 5. Therefore,
the experiment described as follows was conducted to
determine whether succinic acid can be inhibitory to
Pseudomonas.
In a separate experiment, plate count ajar
10 (2.35 gym for 100 ml of water) was prepared, autoclave,
and placed in separate containers and sufficient
succinic acid was added to produce plates which
contained either 0, .25, .5, .75, 1, 2, 3, or I of
succinic acid. The pi was adjusted to 4.7 for one set
of plates; and the pi was adjusted to 7.0 for another
set of plates. Then, each set of plates was inoculated
with either Pseudomonas fluoresces or Pseudomonas
alcaligenes, and these plates were incubated at room
temperature for 24 to 48 hours.
The plates inoculated with Pseudomonas
fluoresces at pi 4.7 all became contaminated with mold
and were not evaluated for Pseudomonas growth. Those
plates inoculated with Pseudomonas fluoresces at pi 7.0
showed growth of Pseudomonas fluoresces within 24 hours
when the concentration of succinic acid was I or less.
The 3% and 4% succinic acid plates did not exhibit
growth of Pseudomonas fluoresces in 24 hours but did
become positive for growth at 48 hours.
Of the Pseudomonas alcaliqenes plates, the
control plate and those which contained succinic acid at
pi 7.0, all supported growth of the organism at 24
hours. In addition, the control plate at pi 4.7 was
positive for growth of Pseudomonas alcaliqenes after 48
hours of incubation. However, all plates containing
35 1/2% succinic acid or more and adjusted to pi 4.7 did
not support Pseudomonas alcaliqenes growth after 48
hours of incubation.
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It is clear from the above data that succinic
acid can inhibit the growth of Pseudomonas alcaliqenes
at pi 4.7 in fairly low concentrations but is not very
active at pi 7Ø The data also demonstrate that
Pseudomonas alcaliqenes is not as susceptible to the
effects of succinic acid at pi 7 as is Pseudomonas
fluoresces.
2. Prop ionic Acid
Although prop ionic acid is known to have
certain valuable abilities as an inhibitor of microbial
growth, it was discovered by experimentation that there
are additional, previously unexpected uses for
preappoints.
a. Bacteria
It was found that preappoints are quite
effective to inhibit Pseudomonas bacteria.
EXAMPLE 3
In order to further investigate the effects of
pure succinic acid and pure prop ionic acid against
Pseudomonas fluoresces and Pseudomonas alcaliqenes, the
following experiment, wherein the pi of the growth media
were adjusted to 5.2, was conducted. The pi of 5.2 was
chosen because this is about the highest pi which one is
likely to encounter in good cottage cheese. Plate count
ajar was prepared as previously described and adjusted
to contain 0.5, 0.75, 1, 2, or 3% of succinic acid, and
separately prepared was plate count ajar containing
0.5%, 0.75%, I 2%, 3%, and 4%, of prop ionic acid. The
final pi was adjusted to 5.2 for all of the media; and
the plates were allowed to dry and inoculated with 0.2
ml of either Pseudomonas fluoresces or Pseudomonas
alcaliqenes cultures. The plates were then incubated at
room temperature for 48 hours. The Pseudomonas
alcaliqenes control plates showed growth in 36 hours;
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and the Pseudomonas fluoresces control plates supported
growth within 48 hours.
The only concentration of succinic acid which
inhibited growth of any pseudomonas was the 3%
concentration of succinic acid which did inhibit the
growth of Pseudomonas fluoresces. However, all other
concentrations of succinic acid allowed growth of
Pseudomonas fluoresces and Pseudomonas alcaliqenes.
The Pseudomonas alcaliqenes and Pseudomonas
fluoresces were both inhibited by all concentrations of
prop ionic acid, including the lowest concentration
tested which was 0.5%.
b. Yeast
Perhaps the most surprising discovery, so
contrary to conventional wisdom, is that preappoints are
effective in inhibiting yeast in food products over a
substantial period of time and that the concentration of
preappoint ions can be sufficiently low that there is no
adverse effect on flavor or aroma of even a delicate
food. This discovery is of particularly great value in
the preservation of yogurt wherein yeast contamination
is a substantial problem. However preappoint ions from
added prop ionic acid or preappoint salts, should be
effective when mixed into any bendable food product.
Although the effect of pi on yeast inhibition by
preappoints has not been fully explored, it appears that
preappoints are effective at oh's below 5.2 and clearly
so at pi 3.35 and below.
Prop ionic acid in concentrations of 0.2% has
been reported to inhibit mold (Handbook of Food
Additives, Second Edition, CRC Press, 1972, pages
137-141). However, prop ionic acid has been reported not
to inhibit yeast and, in fact, concentrations of 0.3%
35 are used in the manufacture of bread or rolls to act as
an inhibitor of mold, but the yeast which is used for
production of bread or rolls is not inhibited.
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In order to preserve yogurt or other similar
food products, a nontoxic source of preappoint ions is
provided and the pi adjusted, if necessary, so that the
desired degree of yeast inhibition is achieved. The
optimum preappoint concentration and pi will vary
somewhat depending on the composition of the food
product, but can be determined by experimentation.
With plain yogurt, the preappoint source is
best added before fermentation to avoid any blending of
the yogurt thereafter. The preappoint source may be
added at any time to mixed, eye. fruit-flavored, yogurts
and many other food products.
EXAMPLE 4
15 Yogurt spoilage occurs both through
contamination of mold and yeast. Therefore, the
following experiment was conducted in order to determine
if prop ionic acid would inhibit mold and/or yeast which
develops in yogurt. Commercially available yogurts
(Yoplait*strawberry, apple, and plain) were purchased
from a grocery store and maintained at room temperature
for several days in order to allow the mold and yeast
present to develop. The mold and yeasts which developed
in these yogurt samples were isolated on plates and
allowed to develop. They were then maintained
separately as "pure" cultures of either mold or yeast.
Potato dextrose ajar (3.5 g of potato dextrose
ajar powder and 0.85 9 of tartaric acid in 100 ml of
water) was prepared, autoclave, and mixed with
prop ionic acid to produce either 0.25%, 0.5% or 0.75~
prop ionic acid. The control plates with no prop ionic
acid had a pi of 3.6 and the plates containing prop ionic
acid had a pi of between 3.25 and 3.35. Therefore,
control plates with no prop ionic acid were Allah prepared
35 with addition of hydrochloric acid so they were
available at pi 2.1 and 2.8. After all plates were
prepared, the mold and yeast which had developed in the
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commercially purchased yogurt was transferred to each
plate and the plates were incubated at 33C for 48 hours
and then maintained at room temperature.
Those control plates with 0% prop ionic acid at
pi 3.6 had yeast colonies present after 36 hours and
mold developed after 2 days at room temperature. Those
control plates containing added hydrochloric acid (pi
2.8) also supported the growth of yeast and mold after
48 hours at room temperature; and a slide of the
organisms confirmed that yeast was growing. The control
plate at pi 2.1 supported the growth of mold only after
7 days and did not support the growth of yeast at this
low phi
After 7 days, the plates containing all
concentrations of prop ionic acid did not support any
growth of either mold or yeast. It is clear from the
above data that prop ionic acid in a concentration as low
as 0.25 percent inhibits the growth of both mold and
yeast strains which contaminate commercially available
dairy products. The results also indicate that
preappoints are effective mold and yeast inhibitors at
concentrations sufficiently low that an effective amount
of such substances would not adversely affect the flavor
or aroma of even a delicate food product. Such results
are not taught by the prior literature, since usual
concentrations of prop ionic acid which inhibit mold are
0.2%. Furthermore, the baking industry routinely uses
calcium preappoint which is not inhibitory to the growth
of yeast at 0.31% (Handbook of Food Additives, Second
Edition, CRC Press, 1972, pages 137-141).
B. MIXED METABOLIZES OF PROPIONIBACTERIA
A further discovery is that Propionibacterium
cultures can be used to produce a growth mixture,
including metabolizes, that inhibits mold, yeast, and
certain bacteria in any of a wide variety of food
products. Some inhibition of spoilage microbes is
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probably due to the presence of prop ionic acid as a
metabolize of propionibacteria. But, the degree of
inhibition achieved is much greater than would be
expected for the amount of prop ionic acid in the
metabolizes. This indicates that an unidentified
substance or substances in the growth mixture is
responsible for the excellent ability of the growth
mixture to extend the shelf life of food products.
As mentioned above, viable propionibacteria are
used in the manufacture of Swiss cheese to form eyes by
the production of COY and to impart the characteristic
Swiss cheese flavor. And, it is now discovered that
there are advantages to adding viable propionibacteria
directly into a batch for other cultured dairy
products. In most food products, however, the presence
of viable propionibacteria would be unacceptable because
eyes would not be desired and a COY release would
bloat packaging materials. Thus, as described in
certain of the following examples, propane- bacteria
can also be grown in a liquid growth medium which is
subsequently heated or otherwise treated to render the
bacteria not viable. The result is a stable mixture
which is an effective additive for the inhibition of
spoilage bacteria in food products.
To facilitate storage and shipping, a
propionibacteria growth mixture may be frozen or
concentrated, e.g. by spray-drying, or freeze-drying, to
form a powder.
A growth mixture according to the present
invention is most readily used by mixing with a
bendable food product, but should also be effective to
treat the surface of solid food products, or the
interior of such products, e.g. by injection. The
optimum amount to be used will depend on the composition
35 of the particular food product to be treated, but can be
determined by simple experimentation.
In most instances, substantial improvements in
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shelf life can be obtained by adding the growth mixture
in an amount sufficiently small that it will have no
deleterious effect on the flavor or aroma of the food
product. The growth mixture includes the numerous
metabolizes of propionibacteria, more than one of which
metabolizes appear to be active in microbial
inhibition. But, as an indication of the quantity of
total metabolizes in a growth mixture, it is convenient
to refer to the amount of preappoint radical present.
Typically, a growth mixture will be effective when mixed
with a bendable food product if the mixture is present
in an amount sufficient that the preappoint radical
contributed by the growth mixture is from 5xlO 7 to
0.1 weight percent of the food product.
Example 5 illustrates, generally, the
effectiveness of propionibacteria growth mixtures.
EXAMPLE 5
Six different strains of ProPionibacterium were
grown in a commercially available (PHASE 4,
~alloway-West Co., Fond Du lag, Wisconsin) bulk starter
medium wherein 39 g of the medium was added to 500 ml of
water, pasteurized at 85C for 45 minutes, cooled to
30C, and then inoculated with 10 ml of 6 different
cultures grown in tomato juice media for 24 hours. The
ProPionibacterium cultures tested were: p-31-c 13673;
8262; 9615; 9616; 9617. These were all obtained from
the American Type Culture Collection in Rockville,
Maryland.
After times 0, 16, 18, 21, and 24 hours, 25 ml
of the growth media were removed by pipette, mixed with
25 ml of double strength potato dextrose ajar (39 g for
500 ml of water) and the mixture was autoclave. After
the potato dextrose ajar and sample were mixed, the
35 sample was brought down to pi 3.0 to 3.7 with
hydrochloride acid and poured into plates. The plates
were dried overnight and then mold or yeast was streaked
,
.~. ...
~Z~8~39~
-- 19 --
onto each plate the next day and the plates were
incubated at 33C for 24 hours. Plates containing the
zero hour propionibacteria growth medium showed a large
amount of growth of mold and yeast. The samples
collected at 16, 18, 21, and 24 hours all allowed the
growth of yeast (most after 24 hours) but mold was
inhibited on these plates, even though the samples
collected and used for zero hour plates did have
extensive mold growth.
For p-31-c, yeast was apparent after 20 hours
and there was no mold for 60 hours. For 13673, yeast
was present after 24 hours and no mold was apparent
after 72 hours. For 8262, there was both yeast and mold
after 24 hours. For strains 9615, 9616, and 9617, yeast
was present after 24 hours; but no mold appeared during
72 hours of observation. Samples of growth media
containing 9617 were somewhat inhibitory to yeast in
this experiment as indicated by less growth of yeast on
plates containing samples of 9617.
Duplicates of the above plates were prepared
after continuing to grow the propionibacteria from 24 up
to 80 hours and utilizing samples collected at times 0,
16, 18, 21, 24, and 80 hours after beginning of growth.
Each plate was divided into 3 parts and 1 part was
streaked with mold and the other two parts were each
streaked with 2 different kinds of yeast. The results
for these duplicate samples showed that for strain
p-31-c, none of the samples prevented complete growth of
yeast or mold, except the 18-hour sample which prevented
mold growth. For strain 13673, the 80-hour sample was
inhibitory to mold, but none of these samples prevented
yeast or mold growth. For strain 8262, none of these
samples were inhibitory to mold or yeast. For strain
9615, the 24-hour sample and the 80-hour sample were
inhibitory to mold and the 80-hour sample was inhibitory
to yeast. For strain 9616, the 80-hour sample was
inhibitory to mold and slightly inhibitory to yeast.
*, .,
lZ~8~4
- 20 -
For strain 9617, the 24-hour and 80-hour samples were
inhibitory to yeast and the 18, 21, 24 and 80-hour
samples were all inhibitory to mold.
These data show that strains 9615, 9616, 9617,
and 13673 which have been allowed to grow for 80 hours
under the conditions described herein can inhibit growth
of yeast or both yeast and mold, and that strains 9616
and 9617 were most active in inhibiting yeast and mold.
An additional experiment was conducted wherein
each of the cultures were sampled 18 hours after growth
and 100 hours after growth and the samples which were
collected were 1 ml and 5 ml and 10 ml of the growth
medium at 18 hours and 1 ml and 5 ml at 100 hours.
These samples were then mixed with 20 ml of potato
dextrose ajar (58.5 g for 1000 ml, autoclave and poured
into plates after adjusting the pi to between 3.0 and
3.6 using concentrated hydrochloric acid). The plates
were dried overnight and inoculated as spread plates
with 0.2 ml of each of 2 different kinds of yeast or
with mold. Plates were then incubated at 30C for 30
hours. Results showed that 10 ml of strain 9615 was
inhibitory to mold after 18 hours of growth and 10 ml of
strain 9617 was inhibitory to mold after 18 hours of
growth. The other samples were not inhibitory after
only 18 hours of growth. However, after 100 hours of
growth, strain 13673 was inhibitory to mold and yeast
for the S-ml sample and to mold with the l-ml sample.
Strain p-31-c was not inhibitory. Strain 8262 was
inhibitory to mold with the 5-ml sample only. Strain
9615 was inhibitory to mold with the l-ml sample and
inhibitory to both mold and yeast with the 5-ml
samples. Strain 9616 was inhibitory to mold with the
l-ml sample and inhibitory to yeast and mold with the
5-ml sample. Strain 9617 was inhibitory to mold with
35 the l-ml sample and inhibitory to both yeast and mold
with the 5-ml sample. It should be noted that the most
inhibitory to mold of all these strains was 9616 where 1
A
94
- 21 -
ml of the growth medium inhibited the growth of molds on
the plates for over 75 hours, which is longer than any
of the other growth media inhibited mold at such a low
concentration. Control plates showed the presence of
mold and yeast after only 30 hours of incubation.
As mentioned above, growth mixtures according
to the present invention will perform somewhat
differently in different food products. The following
examples illustrate ways to determine how best to use
lo ProPionibacterium growth mixtures according to the
present invention with specific food products.
1. Yogurt
For the purpose of this disclosure, yogurt is
defined as a fermented milk product made by allowing
Lactobacillus bulgaricus and Streptococcus thermophilus
to grow in pasteurized milk until a firm coagulum is
formed.
Commercial yogurt products typically have a pi
of 3.5 to 4.5 and a titratable acidity in the range of
0.9 to 2.0 percent expressed as lactic acid. Most
commercial yogurts have a fat content of two to four
percent. Yogurt may contain coloring and flavoring
ingredients, including artificial and natural fruit
flavorings, whole fruit and fruit syrups. Yogurt,
particularly the unflavored variety, has a
characteristic flavor and aroma that would be expected
to be compromised if large amounts of preappoints were
added.
It is the expectation of many consumers and a
requirement in many countries that yogurt contain viable
fermentation microorganisms. Such bacteria are consumed
to aid digestion. To retain viable bacteria, yogurt
cannot be given a final pasteurization to preserve the
35 product for shipment and storage. Thus, yogurt is
particularly susceptible to spoilage microorganisms
present at the manufacturing and packaging site. Due to
~Z~8~94
- 22 -
its composition, mold and yeast growth are the most
common spoilage problems.
As demonstrated by the following examples, the
shelf life of yogurt can be extended greatly, without
pasteurization that would harm the fermentation
bacteria, by adding ProPionib-acteri-um to a yogurt batch
before fermentation or by adding a Propionibacterium
growth mixture according to the present invention,
either before or after yogurt fermentation. Quite
surprisingly, it was found that addition of the
propionihacteria or the growth mixture to a
yogurt-making batch had no noticeable detrimental effect
on the activity of the fermentation bacteria.
A number of examples report the addition to
yogurt of a growth mixture wherein Propionibacterium
cells have been made not viable. This is expected to be
the best commercial procedure. But, the use of a growth
mixture with viable Propane bacterium might be
advantageous due to continued production of metabolizes
2Q in the yogurt. In a "Swiss-style" mixed fruit yogurt,
eyes formed by COY released from Propionibacteria
might prove to be a desirable sensory characteristic.
EXAMPLE 6
A commercially available home style yogurt maker
(Salton*yogurt maker) was used to produce cups of yogurt
. The starter cultures employed were commercially
available for the production of yogurt cry. Hansen's
Laboratories, Milwaukee, Wisconsin). The culture used
30 was R6. The commercially available frozen culture was
thawed and added to milk and allowed to mature prior to
use. Then 1 ml of this culture was added to 200 ml of
2% milk and the yogurt making instructions followed.
Excellent yogurt was produced which had a good body, and
35 was a uniform product with a final pi of 4.2. In a
separate yogurt preparation, the inoculum consisted of
,~_ 1/2 ml of commercially available Hansen's culture
* Trade Mark
:1218~394
- 23 -
combined with 3 ml of Propionibacterium culture. An
excellent yogurt was also produced with good body and a
final pi of 3.9. In addition, yogurt was prepared by
mixing 1/2 ml of Hansen's culture with 3 ml of
Propionibacterium in 200 ml of I milk which had been
fortified with 9 q of nonfat dry milk. The final pi was
3.98 and an excellent, uniform body yogurt was
produced. Thus, the presence of the ProPionibacterium
did not inhibit the production of yogurt in any way.
This is quite important since Pro~ionibacterium are
known to produce carbon dioxide which might disrupt
yogurt formation, but this was not observed. A
combination of lactobacillus and propionibacteria bus
been reported to increase carbon dioxide production
("Stimulating Effect of Lactobacilli on the Growth of
Propionibacteria in Cheese," F.F.J. Nieuwenhof, J.
Standhouders and G. Hut, Net. Milk Dairy J. 23, p.
287-239, I969).
- EXAMPLE 7
In this example, yogurt was prepared by
inoculating pasteurized 6.25~ nonfat dry milk in water
with ProPionibacterium~ waiting 12 hours and then
inoculating with commercially available Hansen's*yogurt
culture. The initial inoculum of the Propionibacterium
was either 1, 2, 4, or 10 I of the mature cultures. At
the end of 12 hours, the pi values were, respectively,
5.5, 5.9, 5.2, and 4.2. A small amount of carbon
dioxide was produced during this time period as
determined by visual observation. Each of these media
was then inoculated with 1 ml of Hansen's yogurt culture
and the milk was allowed to incubate for an additional 8
hours. At the end of this time, the pi values of the
samples were, respectively, 4.5, 4.4, 4.5~ and 4.6 and
the viscosity, texture and flavor of the yogurt were
very good.
In addition, an experiment was conducted
,.
* Trade Mark
~Z~8~94
- 24 -
wherein 10 ml of ProPionibacterium was inoculated into
the nonfat dry milk, allowed to incubate for 12 hours,
and then heated to 85C for 40 minutes to kill the
propionibacteria and then cooled to inoculation
temperature and inoculated with Hansen's yogurt
culture. This also produced an excellent yogurt with
very high viscosity and smooth, fine texture. Thus, it
has been demonstrated here that in the production of
yogurt, the milk could be preincubated with
propionibacteria and then inoculated with yogurt
producing cultures either with or without pasteurization
in order to kill the propionibacteria, and an excellent
quality yogurt can be produced.
Since propionibacteria are known to grow slowly
at refrigeration temperature and to produce prop ionic
acid under these conditions (Hutting and Reinhold, The
Propionic-Acid Bacteria-A Review, J. Milk Food Tuitional.
Vol. 35, No. 5, pp. 295-30; 1972) this example teaches
that the propionibacteria can be grown to substantial
numbers in milk and then yogurt cultures can be added to
produce yogurt, and then the product could be stored in
the refrigerator and, with time, viable propionibacteria
could produce prop ionic acid or some metabolizes which
would be inhibitory to the development of mold and yeast
or some bacteria as taught elsewhere herein.
A similar effect could be obtained by
inoculating yogurt milk with large numbers of
Propionibacterium cells immediately prior to inoculation
with yogurt bacteria. The large number of
I propionibacteria might be obtained either by
concentration or growing to large numbers with internal
or external naturalization of the culture media. There
is no teaching in the prior art that the
propionibacteria can be used either by preincubation or
by simultaneous inoculation with yogurt producing
bacteria in order to produce a good quality yogurt which
would be inhibitory to mold and yeast growth.
. .
SLY
- 25 -
EXAMPLE 8
Yogurt was prepared by pasteurizing 2% milk at
90C for 5 minutes, cooling to 30C and pouring 200 ml
each into different autoclave clear, plastic, 8 ox wide
mouth jars with screw top caps. Nonfat dry milk powder
(9 gym) and sucrose (11.5 gym) were added to each cup and
mixed well by shaking.
Each cup was placed in a constant temperature
water bath (42-44C) and sterile, disposable pipes
were used to inoculate with cultures as shown in the
following tables. The screw-top caps were tightened,
container contents mixed well by shaking, and the water
bath temperature adjusted to 35C i 0.5C, and the
inoculated samples were incubated overnight.
Samples were collected the next morning and
allowed to stand undisturbed, without opening the caps,
at room temperature or in the refrigerator as indicated
in the following tables. Each sample was visually
inspected (through the clear plastic cup) periodically
for the development of mold, sample viscosity and gas
formation as evidenced by bubble or "eye" formation.
The results are given in Tables I and II.
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-- 26 --
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The data in Table I show that the inclusion of
propionibacteria along with usual yogurt-producing
organisms allows production of a good consistency yogurt
which can retain a good consistency if there is an
appropriate balance in the amount of each culture used.
Those samples which developed gas bubbles or "eyes" may
be desirable products if the gas production is not
excessive since the appearance is not unpleasant and
might be useful in marketing 'iSwiss-style" yogurt.
The room temperature data also show that mold
developed in 7 days in the control sample (A-7) but was
delayed by incorporation of propionibacteria until the
Thea day or longer. This "doubling" of the "shelf-life"
is a substantial improvement.
I 4
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- 31 -
The data in Table II confirm the data in Table
I in that the presence of propionibacteria increase
substantially the length of time prior to appearance of
mold; also the growth of mold, once it does appear, is
much more rapid or "heavier" in the controls without
propionibacteria.
Gas chromotographic data indicate that no much
prop ionic acid was produced in these products. The data
are shown below
Sample Identifi-Parts per Million
ligation Number of Prop ionic Acid
Aye Controls
B-7 7.9
A-2 6.9
A-4 9.5
B-2 16.7
This example teaches that propionibacteria can
be incorporated in the production of yogurt and will
extend the shelf-life of the yogurt.
EXAMPLE 9
Propionibacterium Sherman (ATTICS Strain 9617)
was grown in 800 ml of water containing either 62.4 g of
a commercially available bulk starter medium (Formula
l-Phase 4, Galloway West Company, Fun Du lag, Wisconsin)
or in a formula (formula 2) which contained in 800 ml of
water, whey (28.8 g), yeast (4.0 g), diammonium
phosphate (10 g), and citric acid MindWrite (1 g).
Each formula was pasteurized at 85C for 45 minutes, and
then cooled to 31C and inoculated with 10 ml of
Propionibacterium Sherman 9617 which had been grown
for 20 hours at 31C in milk. The organisms were
allowed to incubate at 30C for 90 hours and at that
time 1 ml and 5 ml samples of the growth media were
transferred from each of the flasks and separately mixed
with 20 ml of potato dextrose ajar (29.25 g of potato
dextrose ajar in 500 ml of water) and the pi was reduced
,:
~L8~39~
- 32 -
to 3.1 to 3.7 before pouring the plates. Three plates
were made for each dilution and they were dried
overnight at room temperature, inoculated 24 hours later
with 0.2 ml of mold and 0.2 ml each of two different
types of yeast. Yeasts and mold had been isolated from
commercially available yogurt. These plates were then
incubated at 31C for 48 hours. The results were the
same for the two formulae tested in that 1 ml of the
growth media did not inhibit yeast or mold and 5 ml of
the growth media did not inhibit yeast but did inhibit
mold growth. After 5 days, there was no growth of mold
on any of the plates which had been inoculated with 5 ml
of the growth media. It should be noted that control
plates supported growth of mold and yeast after only 30
hours.
The above growth media which had been allowed
to grow for 90 hours were allowed to continue to grow
for an additional 10 hours and then the pi was raised to
between 7.5 and 8.0 with a slurry of calcium hydroxide
and the 800 ml of growth media for each formula was
divided into two 400 ml portions and dried two different
ways. The first 400 ml portion of each growth medium
was evaporated to semi dryness using a rotary evaporator
and a water vacuum at a temperature of about 70C and
then the powder was placed in a vacuum oven at 80C and
dried; finally the powder was pulverized using a
hammer mill. The initially light cream colored growth
medium produced quite a dark brown powder.
A quantity (300 ml) of each formula (1 and 2
above) was lyophilized by freeze-drying after 100 hours
of growth. This produced a creamy white powder.
Powder (5 g of each of the above dried powders)
was added to 50 ml of water and 3.9 g of potato dextrose
ajar was added to 50 ml of water and the liquids were
autoclave separately, mixed together, and the pi was
lowered to 3.6 using hydrochloric acid before the plates
were poured. Plates were then allowed to dry overnight
I.. .:~
12~B894
- 33 -
at room temperature. Three plates were made for each
powder and 0.2 ml of yeast (two separate types) and 0.2
ml of mold were each transferred to each plate as a
spread plate. The mold and yeast had been isolated from
a commercially available contaminated yogurt sample as
one loopful of culture which was transferred to 5 ml of
sterile water which was vortexes well and then
transferred as a 0.2 ml sample to each plate. Plates
were then incubated at 31C for 40 hours. The results
lo were quite spectacular in that there was no growth of
either type of yeast or of mold on any of the plates
which contained either the freeze-dried or the heat
dried powder. However, there was extensive mold and
yeast growth on the control plates.
Yogurt was prepared by using 200 ml of I milk
and following the usual procedures for preparation of
yogurt with inoculation of 0.5 ml of
commercially-available Hansen's yogurt producing
bacteria. The incubation temperature was 35C for 17
hours. The control sample was fortified with 9 g of
whey and 11.5 g of sucrose; 20% of a commercially
available strawberry fruit flavor was added after the
yogurt was formed. A "plain" control was also prepared
which did not have the added fruit flavor. In addition,
a control fortified with 9 g of nonfat dry milk and 11.5
g of sucrose with added fruit Flavor was also prepared.
The initial pi of each of these preparations was 6.45 to
6.5 and the final pi was 4.15 to 4.4. Each produced a
very smooth, uniform yogurt product with an excellent
consistency and each of these exhibited mold growth on
the surface beginning between the Thea and Thea day after
production of the yogurt.
A yogurt formula was also prepared containing
the same ingredients as the control above except that 9
g of whey was replaced with 7 g of whey plus 2 g of the
heat dried powder produced by growing Propionibacterium
in Formula l. Flavor was also added after formation of
~21~3894
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the yogurt. The initial pi of the milk before formation
of yogurt was 6.5 and the final pi was 4.6. The final
product was discolored because the heat dried powder was
discolored and the product was somewhat separated and
was not uniform in viscosity. Mold growth was inhibited
and did not appear on the surface of this formula until
the Thea day.
An additional yogurt sample was prepared which
contained only 1 g of Formula 1 and 8 g of whey and this
product was very similar to the preceding product in
appearance and the mold did not appear on the surface of
this product until the Thea day. It should also be noted
that there appeared to be some yeast present since
carbon dioxide was slowly evolved from this product.
lo Yogurt was also prepared which contained 6.3 g
of the heat dried powder from Formula 2 identified above
and this product was prepared in 200 ml of 2% milk which
had been fortified with 11.5 g of sucrose. After
preparation, the fruit flavor was added. This product
had an acceptable consistency and did not show any
evidence of mold growth by the Thea day.
The freeze dried powders identified above were
also used to produce yogurt. The control yogurt was
made from 200 ml of 2% milk fortifies with 9 g of whey
and flavor was added after production. In addition, a
control was prepared which was not flavored and a
control was prepared which was fortified with 9 g of
nonfat dry milk and was flavored. All controls produced
excellent products and developed mold growth after the
Thea day of storage at room temperature.
The samples containing the freeze dried powder
were all flavored after production and were all made
from 200 ml of 2% milk. The first sample was fortified
with 4-1/2 g of whey and 4-1/2 g of powder which had
been freeze dried and was produced by growing the
Propionibacterium in a commercially-available medium
(Formula 1). The second sample contained 2 g of the
~Z~8894
- 35 -
same freeze dried powder and 7 g of whey; the third
sample contained 4-1/2 g of the freeze dried powder from
Formula 2 above and 4-1/2 g of whey; the fourth sample
was fortified with 2 g of the freeze dried powder from
Formula 2 above, and 7 g of whey. In all of these
cases, a product with excellent consistency was formed
and there was no apparent yeast or mold growth after 6
days in any of these products treated with the freeze
dried powders. The color was much better than when the
powder had been heat dried although there was some odor
which appeared to be from the whey base growth media in
these final products.
This present example shows that a product can
be produced by growing propionibacterium on whey base
media which, after drying, will be effective inhibiting
yeast and mold growth in dairy products.
It is apparent from other examples herein that
the Propionibacteria can be grown in nonfat dry milk and
a product with a very acceptable odor and color can be
produced after drying the growth media.
EXAMPLE 10
In 800 ml of water was prepared whey (100 g),
yeast extract to g), and diammonium phosphate (8 g) and
this is referred to as Formula 1 for this example.
Formula 2 for this example consisted of whey (100 g),
yeast (5 g), and diammonium phosphate (12 g). Each
formula was pasteurized at 85C for 45 minutes, the
temperature was allowed to cool to 35C, and the growth
media were then inoculated with 10 ml of
Propionibacterium Sherman (10 ml) strain 9617 which
had been grown in sodium lactate broth. These culture
media were allowed to grow for 90 hours and then 5 ml of
each culture was mixed separately with 20 ml of potato
dextrose ajar (19.5 g for 250 ml) and the pi was lowered
to 3.4 using hydrochloric acid before the plates were
poured. Three plates were made for each formula.
j I..
~Z1~3894
- 36 -
Plates were dried overnight and then inoculated with 0.2
ml of either mold or each of two different kinds of
yeast and then incubated for 24 hours at 31C. Yeast
and mold growth appeared on control plates within 36
hours but yeast did not appear on any of the plates
containing the growth media until after 6 days and mold
did not appear on any of the plates containing growth
media for up to lo days.
The culture was allowed to continue to grow for
lo an additional 6 hours or a total of 96 hours and then
the samples were divided and a portion of each growth
medium was freeze dried and a portion was heat dried.
The powders so prepared were mixed (5 g of powder plus
50 ml of water) with potato dextrose ajar (3.9 g of ajar
plus 50 ml of water). It should be noted tot the pi of
the growth cultures was not raised before drying, which
is different from a previous example. Each of the
solutions prepared were autoclave and then the potato
dextrose ajar and the freeze dried or heat dried powder
solutions were combined, the pi of each plate was
adjusted to 3.0 to 3.6 with hydrochloric acid, and the
plates were poured. After drying, each plate was
inoculated with 0.2 ml of yeast or 0.2 ml of mold, and
the plates were incubated for 72 hours at 31C. It
should be noted that the pi of some of the dried powder
plus water solutions was raised to about 7.5 prior to
autoclaving and the pi was not raised up to 7.5 before
autoclaving for some other preparations.
When the pi of the heat-dried powder plus water
mixture was increased prior to autoclaving, there was
complete inhibition of yeast and mold for both Formulas
l and 2 prior to 72 hours and then some mold did appear
on the plates after 72 hours of incubation.
For the freeze-dried powder when the pi was
raised prior to autoclaving, the plates were negative
for yeast and mold for at least 48 hours.
For the freeze dried powder (Formula 2) when
. .
~Z18894
- 37 -
the pi was not raised prior to autoclaving, the plates
were negative for both yeast and mold for 48 hours.
The growth medium which had not been dried was
centrifuged and the supernatant was filtered through a
0.22 micron diameter pore size filter and S ml of the
filtrate for each formula was mixed with potato dextrose
ajar and the plates were inoculated as indicated above.
The filtrates were inhibitory to both yeast and mold as
indicated by a more rapid growth of yeast and mold on
control plates than with those plates which contained
filtrate. After 72 hours, the supernatant from Formula
1 above did allow the growth of yeast and was marginal
with respect to slight growth of mold. However, the
supernatant from Formula 2 completely prevented yeast
and mold grown for the entire 72 hours.
This experiment shows clearly that
propionibacteria growth medium can be converted to a dry
powder either by heat drying or freeze drying and the
powder produced will inhibit yeast and mold. In
addition, this example demonstrates that the growth
medium supernatant liquid contains inhibitory
substance(s) which can be used to inhibit yeast and/or
mold.
EXAMPLE 11
Yogurt was prepared by inoculating 200 ml of 2%
milk fortified with nonfat dry milk (4.5 g) and sugar
(11.5 g) as a basal formula. Commercially available
yogurt cultures (Hansen's) were used.
Propionibacterium (strain 9617) was grown for
96 hours in a mixture of whey (200 g), diammonium
phosphate (12 g), and yeast extract (5 g), in 800 ml of
water (identification symbol p-a) or in a formula of
nonfat dry milk (200 g), diammonium phosphate (12 g),
yeast (5 g), in 800 ml of water (identification p-b).
Each of these growth media were then either freeze dried
or heat dried as described elsewhere herein. The
i. .
~LZ~8~394
- 38 -
following combinations of ingredients were then used to
prepare yogurt. The basal media also initially
contained sodium caseinate (1.0 g) unless specified
otherwise below and in some cases a fruit flavor (10.0
g) was added after the yogurt had been formed. The
following 12 formulae were tested.
1) Basal medium plus p-a (4.5 g) which had been
freeze dried,
2) Basal medium plus p-a (4.5 g) which had been
heat dried,
3) Basal medium plus p-a (4.5 g) which had been
freeze dried and no flavor,
4) Basal medium plus p-a (4.5 g) which had been
heat dried and no flavor,
5) Basal medium plus p-a (4.5 g) which had been
heat dried and no sodium caseinate,
6) Basal medium plus p-a which had been heat
dried, 4.5 g and no sodium caseinate and no
flavor,
7) Basal medium plus p-b which had been freeze
dried, 4.5 g,
8) Basal medium plus p-b which had been heat
dried, 4.5 g,
9) Basal medium plus p-b which had been freeze
dried, 4.5 9 and no flavor,
10) Basal medium plus p-b which had been heat
dried, 4.5 g and no flavor,
11) Basal medium plus p-b which had been heat
dried, 4.5 g and no sodium caseinate,
12) Basal medium plus p-b which had been heat
dried, 4.5 g and no sodium caseinate and no
flavor.
~Z~889~
- 39 -
Each of the above products produced an
excellent yogurt with excellent viscosity and texture.
Those yogurts which had been prepared with p-b often had
a solid or precipitate on the bottom which was readily
dispersed and mixable with the products which contained
flavor since the stirring of the flavor into the yogurt
distributed any solid which had settled to the bottom.
It is possible that the source of the solid was
coagulated proteins which developed during the growth of
ProPionibacterium prior either to freeze drying or heat
drying of the growth medium when nonfat dry milk was
used as part of the nutrient medium, since a layer of
solid or precipitated material did not develop in those
formulae prepared with p-a which employed whey rather
than nonfat dry milk.
The above products were prepared and stirred
and three additional cups of yogurt which were purchased
commercially were placed in identical containers and
stirred and set out with these 12 products for a taste
test by 3 individuals experienced in dairy microbiology
and yogurt production. Combined, these individuals had
50 years experience in working with dairy products and
have been involved in taste panels for judging yogurt.
In general, the products prepared in this example were
judged to be equal to the commercially-purchased
products, and the preferred formulae were numbers 3, 4,
5, and 12 although the three "taste experts" did not
agree on the order of superiority.
These products were kept in the refrigerator
and examined daily. A commercially available flavored
product exhibited large amounts of gas production and a
yeasty odor and flavor 6 days after purchasing. A plain
yogurt which had been purchased showed growth of mold on
the surface after 20 days in the refrigerator. An
additional flavored yogurt which had been purchased also
showed physical separation and had a very bad yeasty
odor. There was no appearance of yeast in the formulas
Lo`'
aye
-- Jo --
1-12 above after 26 days and the first appearance of
mold appeared on the surface of a few samples after 26
days. Most of the samples did not exhibit mold or yeast
growth for a prolonged period although the exact
appearance date of contamination was not recorded in
this experiment.
In a separate experiment with similar
formulations stored at room temperature, mold often
appeared on the surface of control formulations in four
or five days for products which had been flavored but
those which contained 2.25~ of the powdered growth
medium of propionibacteria did not demonstrate mold
growth until almost three times as long (i.e. 12 to 14
days) and in one exceptional case mold appearance was
delayed for over 40 days, even though the container was
opened and checked daily.
2. Cozily
EXAMPLE 12
"Cozily*" it a commercially available
"non cultured yogurt" product containing stabilizers,
carbide rates and protein Chocolate, apricot, and
blueberry flavored Kisses we're obtained commercially;
and each was divided into controls and test samples.
The controls developed gas bubbles at room
temperature after three days and mold developed on the
top of the chocolate and apricot control icily aster
five days, although mold did not appear in the blueberry
product at that time. In addition, the chocolate
flavored control developed mold after 16 days in the
refrigerator and the apricot flavored product developed
mold after 21 days in the refrigerator.
The test samples were incorporated with 2-1/4-
of the powder produced by drying propionibacterium
growth medium as described in Example 9 herein, and the
products were then stored at either room or
refrigeration (2-5C) temperature in a study design
I- * Trade Mar
~218894
- 41 -
which paralleled the controls described in the above
paragraph. After 30 days, there was no mold or yeast in
any of the Kisses products which had the dried, powdered
propionibacteria growth medium added at either room
temperature or refrigeration temperature. This example
further indicates that the materials produced by growing
propionibacterium can be effective at inhibiting both
yeast and mold in dairy products. The appearance, odor
and flavor of these products with the extended
shelf-life were all excellent.
3. Fruit Juice
he shelf life variety of fruit juices and
related products was extended using propionibacteria
according to the present invention
EXAMPLE 13
Apple cider which had been filtered prior to
packaging and another sample which had not been
filtered, grape juice frozen concentrate, concentrated
raspberry yogurt flavor and concentrated cherry yogurt
flavor for use in yogurt were commercially obtained.
Small paper cups were prepared containing about 100 g of
each type of apple cider and of the concentrated grape
juice and of the grape juice after it had been diluted
according to manufacturer's instructions, and of the
cherry yogurt flavor and of the raspberry yogurt flavor.
Five cups of each was prepared with one cup of
each serving as a control. Other cups were inoculated
with yeast and/or mold obtained from commercial yogurt.
A second cup of each food product was inoculated with 1
ml of viable yeast cells only; and a third cup was
inoculated with 1 ml of viable mold only. The fourth
cup was inoculated with 1 ml of viable yeast and I of a
heat-dried powder produced from growing propionibacteria
medium according to Example 9, and the fifth cup was
inoculated with 1 ml of viable mold and 3% of the
~21138~4
- 42 -
powdered propionibacteria growth medium. These were
then stored at room temperature (uncovered). The
following key was used to identify the effects:
1. Apple cider, filtered a. Mold
2. Unfiltered apple cider b. Yeast
3. Diluted grape juice c. Mold plus
ProPionibacterium
growth medium
4. Concentrated grape d. Yeast plus
juice Propionibacterium
growth medium
5. Raspberry yogurt e. Control
flavor
On the second day, cup l-b (apple cider filtered
and inoculated with yeast) developed a yeasty odor and
cup 3-b (diluted grape juice inoculated with yeast) also
developed a yeasty odor. On the third day, cup l-a
developed mold and cup 3-a also developed mold, and cup
4-b (concentrated grape juice inoculated with yeast)
developed yeast On the Thea day cup l-e had mold, and
cup 4-a had mold. However, cups c and d which were the
products inoculated with mold and yeast and the powdered
Propionibacterium growth medium did not have any mold or
yeast even after four days at room temperature as
contrasted to those samples which did not contain the
Propionibacterium growth medium as described above.
This demonstrates a dramatic usefulness of the
propionibacterium growth medium in inhibiting yeast and
mold in a variety of food products.
EXAMPLE 14
P. Sherman ATTICS 9616 was grown in
frozen-reconstituted orange juice fortified with 0.1%
yeast extract for two days at 30C~ A commercial orange
juice concentrate was diluted 50%, 10~, 5% and 1%. The
pi was adjusted to 5.0 with 1.0 molar Noah. Three sets
. .
lZ~8894
- 43 -
of the above dilutions were made. One set was
autoclave, the second set pasteurized, and the third
set received no heat treatment. The three sets were
inoculated with I P. Sherman culture and incubated at
30C for five days. Centrifugation was carried out on
all the three sets and the supernatant was filter
sterilized and used (5%) against four different molds.
Controls were made from the orange juice receiving the
above treatment without P. Sherman inoculation but
inoculated with the different molds.
Results appear in Table III:
,
, .
1218894
-- 44 --
a + + + + + + + +
++++ .++++
two ++++ ++++
v Jo ++++ I++
o lo ++++
N l l + l l l +
1++1 11+1
O 1++1 11+1 .'
.' I fill fill
11+1 +1+1
I ~.1+1 11+1
H O Jo D I I + I
O 11+1 +1+1 .
O
'
e 61~161 Eye I
o o ::
Jo mu l mu :: I
I O C O X e
I: U Lo O 1:: U L. O
o C I C I I lo D
I :
lZ1~
45 -
These results show that propionibacteria grown
in orange juice produce substances inhibitory to molds
and yeast.
4. Cottage Cheese
For the purpose of this disclosure, cottage
cheese is defined as a soft, uncured cheese made by
coagulating pasteurized skim milk and separating the
curd from the non coagulated liquid.
Typically, cottage cheese has at least 20~
solids by weight. There are two common types of cottage
cheese plain and creamed. The latter is raised in fat
content by stirring in cream or a creaming mixture.
US. Government standards require 4% fat content in
creamed cottage cheese, but partially-creamed cottage
cheeses, having a fat content of 0.5 to 2% are also sold
in the United States.
Coagulation may be accomplished by the addition
of lactic acid-producing bacteria, a suitable
milk-clotting enzyme and/or a food grade acid.
Propionibacteria can be incorporated into
cottage cheese with an effect similar to that described
above for yogurt and other food products. The bacteria
can be produced separately from the cottage cheese and
concentrated after growth and prior to utilization or
just grown to high numbers and then added to the cottage
cheese "cream". Alternatively, the cottage cheese
"cream" can be cultured with the propionibacteria prior
to creaming. Further, the propionibacteria can be used
along with usual cultures to "set" the cottage cheese
from the beginning of the cottage cheese manufacture as
in the case of yogurt.
To produce one antimicrobial growth mixture
particularly suitable for use in cottage cheese and
other dairy products, a Propionibacterium culture is
started in any suitable lactate broth to condition the
culture to metabolize lactic acid. The resulting
~Z~88g4
- 46 -
culture is then used to inoculate a much larger volume
of a growth medium that may include as nutritive
ingredients a source of milk protein, or milk protein
derivative, or a milk protein substitute and lactic
acid, for example skim milk acidified to pi 5.0 to 5.5
with lactic acid. A higher initial pi could be used,
but the medium would then be suitable to sustain the
growth of numerous possible contaminating
microorganisms. The inoculated medium is allowed to
grow at a favorable temperature for as long as necessary
to form a growth in the mixture that is active against
spoilage microorganisms.
The immediately following examples and Example
21 below illustrate the usefulness of propionibacteria
growth mixtures in the preservation of cottage cheese
and, particularly, in the inhibition of psychrotrophic
gram negative, slime-producing cottage cheese spoilage
organisms.
EXAMPLE 15
Flasks (100-ml capacity containing magnetic
stirrer bars) of nonfat milk containing 0.1% yeast
extract were pasteurized at 85C for 45 minutes. They
were cooled to 30C and acidified to pi 5.3 with 10%
lactic acid. ProPionibacterium Sherman (ATTICS Strain
9616) was added at the rate of I from a 48-hour old
culture grown up in sodium lactate broth (Tripticase,
10.0 g; yeast extract, 10.0 g; 60% sodium lactate
solution, 16.7 ml; monopotassium phosphate, 0.25 g;
30 manganese sulfate, 0.005 g or 0.5 ml of a 0.1 M
solution; and water, 1000 ml; pi =7.0 before autoclaving
at 121C for 15 minutes) at 30C. Flasks were placed on
a six station magnetic stirrer and slowly agitated
during the entire incubation period.
A control flask uninoculated with the
propionibacteria was similarly treated. Samples were
taken at 24, 48, 72 and 96 hours and assayed for acetic
i I
~2~8~394
- 47 -
and prop ionic acids using a Model AYE Hewlett Packard
gas chromatography equipped with a Model AYE
integrator. Samples were also tested for inhibitory
activity against a psychrotrophic gram negative,
slime-producing cottage cheese spoilage organism
supplied by H. P. Hood, Inc., 56 Roland St., Boston,
Mass. This organism was found by electron microscopy to
contain both coliform (pericricnous flagella) and
pseudomonas (monotrichous flagella) cells. These
organisms are important cottage cheese spoilage bacteria
(Dr. Paul Swenson, H. P. Hood, Inc., personal
communication) and are now maintained in and available
from the Department of Microbiology at Oregon State
University.
The test for inhibition against this spoilage
bacterium was carried out by adding 1%, 2%, I 4%, 5
and 10% (v/v) amounts of autoclave (121C for 15
minutes) P. Sherman 9616 milk culture taken at the
various time intervals of growth (and autoclaving as
obtained or neutralizing to pi 7.0 with calcium
hydroxide prior to autoclaving) to 100 ml of crystal
violet tetrazolium ajar (CUT ajar - Tryptone 5.0 g;
yeast extract, 2.5 g; glucose, 1.0 g; distilled water,
1000 ml; ajar, 15.0 g; adjust to pi 7.1; after
autoclaving at 121C for 15 minutes, add
filter-sterilized crystal violet at 0.001 g per liter
and 2, 3, 5, triphenyltetrazolium chloride at 0.05 g per
liter). Each lot of medium containing the various
concentrations of the autoclave milk culture was then
acidified to pi 5.3 with sterile 10% tartaric acid.
Several dilutions of an overnight lactose broth (Beef
extract, 3.0 g; petunia, 5.0 g; lactose, 5.0 g;
distilled water, 1000 ml; pi 6.8-7.0) culture of the
psychrotrophic spoilage organism were then made and 1.0
ml allocates added to sterile putter plates. About 10 to
15 ml of CUT ajar then was added and the plates
incubated at room temperature (25C) for 48 hours. On
i
. -
.;
~Z18~39~
- I -
this medium the psychrotrophic spoilage organism grew as
large, (l to 5 mm diameter) deep red, glistening
colonies. The amount of inhibition in comparison to
control plates containing no autoclave P. Sherman
culture could then be calculated as follows for each
concentration of culture taken at the different time
intervals:
I Collins on Control - Colonies on Test Plate loo
Colonies on Control
lo Results obtained in analyzing the neutralized
(pi 7.0) samples for acetic acid and prop ionic acids
were as follows:
TABLE IV
Mel (Pam)
ours Acetic Acid Prop ionic Acid
24 869 774
48 439 556
96 767 706
Essentially the same data were obtained for
unneutraliæed samples.
In analyzing the samples for inhibition of the
psychrotrophic, slime-producing cottage cheese spoilage
organism, the following results were obtained:
TABLE V
Percent inhibition of cottage cheese spoilage
organism by various amounts of P. Sherman 9616
milk culture grown in pasteurized* milk.
Culture Added Time Sampled (hours)
(%1 24 48 72 96
O O O O O
l 62 50 lo 96
2 56 lo lo 90
3 42 lo 44 97
4 88 lo 46 98
96 lo 52 97
3510 50 lo 36 97
*Milk pasteurized at 85C for 45 minutes and then
acidified with lactic acid to pi 5.3.
A
lZ~L8~394
- 49 -
In another experiment, the Propionibacterium
she manic 9616 culture was grown under the same
conditions in nonfat milk except the milk was autoclave
and not acidified to pi 5.3 with lactic acid. This was
done because microscopic examination of the culture
grown in the pasteurized (85Cm 45 minutes) nonfat milk
revealed sporeformers emerging by 48-72 hours. In fact,
the counts of heat tolerant forms (survive 60C for 30
minutes) at zero time and at 72 hours were 100 per ml
and 107 per ml, respectively. It was felt this might
confound interpretation of the data in the pasteurized
samples. The following data were obtained with the
autoclave milk grown cultures.
TABLE VI
__ Mel _ m)
Hoarsest c Acid Prop ionic Acid
24 310 106
48 87 35
72 5~2 713
96 788 1064
. _ _ _ __
In this case, we see the maximum acetate and
preappoint production delayed until at least 96 hours in
contrast to the cultures which were acidified with
lactic acid to pi 5.3. This difference apparently is
due to the fact that acetate and preappoint are produced
from lactate which the propionibacteria did not have to
produce in the acidified samples but which they produced
gradually in the non acidified samples.
Data on inhibition of the cottage cheese
spoilage organism are in the following table.
~Z1~3~394
- 50 -
TABLE VII
Percent inhibition of cottage cheese spoilage
organism by various amounts of P. Sherman
culture grown in auto craved* milk.
Culture Added Time Sampled (hours)
(%) _ 48 96_
O O O O
1 36 90 71
2 44 -- 84
3 44 97 90
4 66 -- 91
100 90 93
100 98 92
_ _ . _ . _ . . , _ _
*Milk autoclave 15 minutes at 121C.
If one considers thaw prop ionic and acetic acids
may be responsible for the inhibition seen, the time at
which these acids are maximally produced should agree
with the times at which samples show maximum inhibition
of the spoilage bacteria. This, however, is not the
case. In the case of the pasteurized, acidified growth
culture, maximum acetate and preappoint occurred by 24
hours but maximum inhibition not until at least 48
hours. In case of the autoclave, non acidified milk,
maximum acetate and preappoint occurred at 96 hours but
maximum inhibition of spoilage bacteria at 24 to 48
2 hours. Therefore, these data suggest that some natural
metabolizes other than acetate and preappoint may also
be involved in the inhibition of the cottage cheese
spoilage bacteria.
EXAMPLE 16
Propionibacterium Sherman (ATTICS Strain 9716)
were grown in a sodium lactate solution for 48 hours as
described in Example 15. five hundred gallons of skim
milk were then pasteurized at 190F for 45 minutes, and
subsequently cooled to 86F. The cooled milk was
acidified using 85~ reagent grade lactic acid to a pi ox
5.3 and then inoculated with 0.5% of the
.
12ï8894
- 51 -
ProPionibacterium Sherman culture. The inoculated
milk was slowly agitated during incubation for 98 hours,
and thereafter neutralized with sodium hydroxide to pi
7Ø The neutralized liquid was pasteurized at 1~5
for 20 minutes, cooled to ambient temperature (about
75F), pumped through sterile lines into six-gallon
sterile plastic bags and then frozen.
When thawed, the liquid medium was very active
in inhibiting the growth of the psychorotropic gram
negative, slime-producing cottage cheese spoilage
organism mentioned in Example 15.
EXAMPLE 17
220 gallons of milk fortified with 0.1% yeast
extract were heat-treated at 85C for 45 minutes and
then cooled rapidly
to 30C. 86~ lactic acid (Sigma Grade) was then added
with agitation until the pi was lowered to 5.3. The
milk was then inoculated with a mature culture of 1.25
Propionibacterium Sherman (ATTICS 9716); and the culture
allowed to grow for 48 hours. The final mature growth
mixture (bacterial soup) was pasteurized, a portion
retained as a liquid, and the remainder spray-dried
using a commercial box-type spray dryer. The liquid and
spray dried powders were incorporated into cottage
cheese dressing to evaluate their effect on the keeping
quality of the final creamed cottage cheese. The liquid
and powder were substituted in the control dressing
formulation for whole milk (for liquid) and nonfat dry
milk (for spray dried powder) on a comparable solids
substitution basis. The dressing was formulated at the
upper level of inhibitor addition to be tested and lower
levels were obtained by back blending at specified ratios
with a control dressing. The final inhibitor addition
level was calculated to be the percent of inhibitor on a
solids basis per pound of finished cottage cheese.
A control cottage cheese was made along with all
,
~8894
- 52 -
testing variables. Initially, in order to establish an
effective but not excessive addition level of the
inhibitor and also a reasonable inoculation level of the
surface slime organism, various samples were evaluated
with different inhibitor addition and inoculation
levels. The slime inoculant was mixed in the cottage
cheese at specified counts per pound of cheese. Control
and inhibitor samples, both inoculated and
non inoculated, were packed and sealed with heat sealed
foil lids. Samples were set in storage for evaluation
at 45 and 50 F.
Evaluations indicated a positive inhibitory
effect of the spray-dried powder in test sample versus
control in that the storage time at 45 and 50F was
increased 7 to 10 days before surface slime growth
appeared. Results with liquid which had 8.8% solids
were similar. The percent inhibitor in the finished
product was 0.00% (control) 0.12~, 0.24%, 0.36~ and
0.48%. All samples were inoculated with spoilage
bacteria at 2000/lb. or 8000/lb. along with a
non inoculated set of each inhibitor level as a control.
Results showing the number of days before surface slime
growth appeared in samples from a 4000 lb. batch of
cottage cheese are summarized in Table VIII:
TABLE VIII
Days at 50F
When Spoilage Inoculation level of
Appeared Sample Spoilage Bacteria
16 Control 8000/lb.
23 Control 2000/lb.
23 0.12% Inhibitor 8000/lb.
44 Control Non-inoculated
56 Owls% Inhibitor 2000/lb.
Flavor evaluations indicate that all
3 non-inoculated inhibitor-containing samples were
acceptable after 44 days at 50F.
121~389~
- 53 -
Thus, the inhibitor extended significantly the
number of days at 50F before surface slime growth
appeared.
Further testing of these products was carried
out in cottage cheese as above with the following
conditions:
(a) Control - No inhibitor added.
(b) Spray dried inhibitor addition - 0.30%
lovely.
(c) Spray dried inhibitor addition -
reconstituted and pasteurized at 255F - 36
sec. - 0.30~ level.
(d) Skim based liquid - 14.50~ solids - 0.15%
level.
Condition (c) was set up to safeguard against
possible microbial contamination by the inhibitor
powder. Condition (d) was included to compare the
inhibitory activity of the liquid product versus the
spray dried sample.
The following table shows results of these tests
where the number of days before the appearance of
spoilage slime is given:
TABLE IX
Days When Conditions
Spoilage Appeared
6 days Control - Inoculated
14 days Condition (b) - Inoculated - 1 of
9 samples
17 days Condition (b) - 100%
Condition (c) - 100
From these observations it is clear that the
spray dried material also displays inhibitory activity
I
sly
- 54 -
but it appears somewhat less active than the liquid
samples, especially since samples under condition (d)
showed no surface growth.
5. Other Food Products
The foregoing examples describe some specific
applications of the present invention. It is
anticipated that propionibacteria growth mixtures will
prove to be safe and effective for use in the
preservation of other food products. Results similar to
those obtained for yogurt and cottage cheese are
expected for the use of propionibacteria metabolizes to
preserve such dairy products as fluid milk, half and
half, whipping cream, sour cream, and the like wherein
psychotropic bacteria are a common spoilage organism.
The metabolizes should also be particularly suitable for
use in the preservation of bendable food products such
as ground meat or in sausages.
It is also anticipated that such growth mixtures
can be applied in liquid form to solid food products
such as fresh fruits and vegetables. Application could
be by spraying, immersion, or injection.
C. SEPARATED METABOLIZES OF PROPIONIBACTERIA
As discussed above in Example 10, the
supernatant liquid of a propionibacteria liquid growth
mixture was effective in inhibiting spoilage
microorganisms. It has been further discovered that the
supernatant is the most effective fraction in inhibiting
mold and yeast. And, surprisingly, it is now found that
solids separated from the supernatant actually stimulate
microbial growth. It is anticipated that the solids
fraction can be added to growth media and the media then
used for an accelerated growth of such commercial useful
microorganisms as Streptococcus lactic, Streptococcus
creamers, Lactobacillus bul~aricus, Streptococcus
thermophilus, Lactobacillus acidoPhilus~ Leuconostoc
Jo
~X18~94
- 55 -
species, Lactobacillus planters, and Pedro coccus
cerevisiae.
The unique properties of propionibacteria growth
mixture fractions are illustrated by the following
examples.
EXAMPLE 18
Forty liters of milk fortified with 0.1% yeast
extract were heat-treated at 85C for 45 minutes in a
Fermacell fermenter model FG-50. The milk was cooled
rapidly to 30C and then acid-treated with 86~ lactic
acid (Sigma Grade) to pi 5.3. The milk was then
inoculated with 2% of a 48 hour-old culture of
Propionibacterium Sherman (Strain ATTICS 9616). After
four days, samples were taken aseptically and placed in
a sterile flask. Two 500-ml amounts of culture were
centrifuged at 10,000 rum for 10 minutes. The
supernatant was placed into sterile bottles while the
sediment was centrifuged and washed three times with
distilled water. One 250-ml portion of supernatant was
neutralized with 1.0 molar Noah to pi 7.0 and the other
250-ml portion was used unneutralized. The clean
sediment and the two portions of supernatant were left
at room temperature. In two days, the sediment
developed a sharp putrid odor and was obviously
spoiled. The supernatant fractions, on the other hand,
were clear and fresh and smelled like Swiss cheese.
They have remained fresh and clear for over three
months. The two supernatants showed no sign of
contamination. This reveals the wide range of
inhibitory activity residing in the supernatant portion
in contrast to the sediment which rapidly spoiled.
Fractionation of such inhibitory activity in this manner
was unexpected.
EXAMPLE 19
Ten percent of the two portions of the
,
1~113894
- 56 -
supernatant material from Example 18 was added to an
acidified malt extract ajar pi 3.5 (bacto-malt Extract
B-186) and poured onto plates. The plates were allowed
to solidify and dry overnight and 0.2 ml of seven
different molds (Asperqillus nicer, Fusarium oxysporum,
Respace species, Mocker species, yogurt mold, cottage
cheese mold, and Penicillium rcqueforti) were spread
over the ajar surface. The controls were made with only
acidified malt ajar inoculated with the molds. Ten
percent of the sediment was subjected to the same
treatment. In two days the controls showed nominal mold
growth while the plates containing the sediment were
very heavily overgrown with the molds. In the
supernatant-containing plates, six of the molds were
totally inhibited while P. roqueforti was only partially
inhibited. The sediment clearly stimulated mold growth
so these plates had more growth than the controls.
The same experiment was done using two yeasts
tKluYveromyces fragile and a yeast isolated from
commercially available orange juice). A pronounced
inhibition by the supernatant material was seen again in
this case. This inhibition by a culture fraction of
Propane bacterium, especially of yeasts, and the
stimulant effect of the sediment was unexpected.
EXAMPLE 20
In experiments to test for inhibition of a gram
negative cottage cheese spoilage organism (slime
producer) supplied by H. P. Hood, Inc., 56 Roland St.,
Boston, Massachusetts, lactose broth (Disco) was
acidified with 10% tartaric acid to pi 5.0 and dispensed
into test tubes for sterilization at 121C for 15
minutes. The supernatant material from Example 18 was
added to two sets of test tubes containing the lactose
broth to make final concentrations of 40%, 20%, 10%, 5%,
2.5%, and 1.2%. One set was inoculated wit 0.2 ml of
the slime producer and the other was not inoculated. A
A
~Z18894
- 57 -
control was 10 ml of lactose broth (pi 5.0) inoculated
with slime producer. All the test tubes were incubated
at 30C for 24 hours. Turbimeteric readings were taken
on all the test tubes at 600 no with a Perkin-Elmer
spectrophotometer model 35.
Results were as follows:
do
~2~88~4
-- 58
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Jo ..
US
TV o o Al or us . C
. Q VOW O O I N (I') Lo
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v vow o I o or v
v z duo o o o o o E
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~Z~889~
59 .
This example shows, therefore, that the natural
metabolizes of P. Sherman will inhibit gram negative
psychrotrophic bacterium which spoil refrigerated food
such as cottage cheese.
Graphs showing these results appear as Figs. 1
and 2. Fig. 1 shows inhibition of gram negative
psychrotrophic cottage cheese spoilage bacterium by 4
day-old neutralized ON P. Sherman supernatant
(inhibitor). Fig. 2 shows inhibition of gram negative
psychrotrophic cottage cheese spoilage bacterium by 4
day-old unneutralized (4/U) P. Sherman supernatant
(inhibitor).
EXAMPLE 21
Milk (220 gallons) fortified with 0.1% yeast
extract in a closed vat at a commercial dairy center in
Port land, Oregon, was inoculated with 1.25~
Propane bacterium Sherman (ATTICS 9616) culture after
being treated the same way as in the Example 18.
Samples were taken at 48 hours and treated with rennet
(1/1000) to precipitate the proteins. Centrifugation
was carried out at 5,000 rum for 5 minutes. The
supernatant was filtered on a Whitman No. 1 filter
paper. The filtrate was then neutralized to pi 7.0 with
1.0 molar Noah.
Two gram negative organisms, the cottage cheese
slime producer of Example 15, and a gram negative
isolate from raw goat's milk, were tested as before for
the inhibitory effect of the filtered supernatant in
lactose broth preadjusted to pi 5Ø Final
concentrations of the inhibitor were 20%, 15~, 10%, 5%,
2.5% and 1.2%. Results are summarized in Tables XI and
XII.
~LZ1~894
- 6 0
Twill I
tnhlbltloo of the sly producer (SUP).
Z lnhlbltorSP I I Difference Z lnhlbl~lon
20'. 0.~440.878 0.066 92%
15% owe 0.069 92Z
10% 0.8630.667 0.196 80%
5% 0.9500.547 0.403 51%
2.5~ 0.9350.439 0.496 40~
1.2;: 0.8610.371 0.490 40;:
Control a. 0.830
T~BLB XII
Inhibition of rho goat r~llk solace (GO).
Z lnl-lbl~or GO + I I Difference X lnhlbltlon
_ _
20Z 0.917 OBOE 0.038 89,:
15,. 0.812 0.774 0.038 80%
lo 0.718 0.665 0.053 70%
5Z 0.574 0.512 0.062 67Z
2.5,. 0.526 0.457 0.069 63Z
1.2% 0.490 0.372 0.118 37%
Control a. 0.186
~Z~8894
- 6] -
These results appear in graphic form in Figs. 3
and 4. Fig 3 is a graph showing inhibition of gram
negative psychrotrophic cottage cheese spoilage
bacterium by supernatant of 2-day culture of P.
Sherman (inhibitor). Fig. 4 is a graph showing
inhibition of gram negative bacterium isolated from raw
goat's milk by 2 day-old supernatant of P. Sherman.
While we have described and given examples of
preferred embodiments of our inventions, it will be
apparent to those skilled in the art that changes and
modifications may be made without departing from our
inventions in their broader aspects. We therefore
intend the appended claims to cover all such changes and
modifications as fall within the true spirit and scope
of our inventions.
A
