Note: Descriptions are shown in the official language in which they were submitted.
~L2~S339
MET~IOD OF G~OWING CHEESE START~R ~lICR ORGANISMS
Back~round of the Invention
-
1. Field of the Invention
5The present invention is broadly concerned
with an improved method of growing or culturing acid-
producing ~icroorganisms used in cheese making. More
particularly, it is concerne~ with an irnproved method
which involves initially permitting an inocu~ated
10medium to incubate until the pH of the medi.um drops to
an appro~riate level, followed by raisinq of the rH and
further incubation to completion. The method has been
shown to gi~Je enhanced results, particularly in con-
junction with a new lecithin-ccntaining starter medium.
152. Descri~tion of the Prior Art
In the manufacture of natural cheese, milk in
a cheese vat is inoculated with a minor amount (e.g.,
2-4 percent) of a bulk starter ~roviding the necessary
culture of acid-forrr.ing microorganisms uscd for the
20particular cheese being manufactured. ~or example, in
the case of Italian cheeses such as mozzarella, it is
the usual practice to employ Streptococcus thermophilus
together with one or more lacto~acilli such as Lacto-
bacillus bulgarj.s. In the art, the stre~tococci are
25generally referred by the short name of "coccus", while
the lactobacilli are referred to as "rod" bacteria
because of their appearance under microscopic examj.-
nation.
The quantity and activity of cheese-making
30microorganisms can be critical to the overall outcome
of the process and final cheese quality. Again refer-
ring to the Ita~.ian cheese, it has been found that, in
order to make acceptable cheese, the ratio of coccus to
rod organisms in the starters shou~d be from about l:l
35to 5:1, the most preferable leve~ beina a~out 4:1. ~f
~2~5339
1these ratio considerations are not met, the final
cheese product may be deficient in flavor or physical
properties such as elasticity and "stringiness."
It is the universal practice among cheese
makers to -grow their hul~ starters using relatively
minor amounts of seed culture. In such techniques, the
seed culture is inoculated into a starter medium,
and allowed to incubate therein so that the culture
cells will multiply to produce the desired bulk starter
for use in cheese makin~. Here again, the types of
starter media and the techniques used during the incu-
hation ~rocess can have a relatively critical outcome
on the qùality of the final bulk starter, and hence cn
the cheese ultimately produced. A dilute dispersion of
nonfat mi~k (e.g., 12 percent solids level) in water
has lona been considered the starter medium of choice.
flowever, use of nonfat milk in this context is a rela-
tively expensive ~roposition, and therefore cheese
makers have in the past sought to use media of a less
expensive nature which either eliminate nonfat milk
entirely, or sharply limit its use by provision of
suhstitute materials. Many of these propcsed media
include constituents such as whey or the like. Exem-
plary patents disclosing prior starter media include
25U.S. Patents ~os. 3,998,700, 2,805,950, and 3,852,158.
The longstanding technique of starter incu-
bation used by cheese makers has been to simply inocu-
late the medium (which is typically at or around neu-
tral pH), while maintaining the medium in a heated
30condition (e.g., 102 degrees F.). During the incu~a-
tion process the seed culture multiplies and produces
acid; this in turn serves to drop the pH level of the
medium down to a level of 4.0-5.0, at which time the t-
itratable acidity of the medium is typically at an
35appropriate level and the incubation is then terminated
12~S33~
1 by coolinq to 40-50 degrees Fahrenheit. While this is
t~e customary approach, workers in the art have devised
a number of different o~erational methods which can, in
certain circumstances, produce more or better ~uality
cheese-makina microorganisms. For example, research-
ers at the Utah State University have developed a
lactic culture system which involves continuous neutra-
lization of the starter medium during incubation.
Specifically, use of this system involves a pH control
system including pH probes, and means for injecting a
base such as ammcnia into the starter medium. Gener-
ally speaking, the starting p~l of the medium is around
6.3, and, as the pH drops during incubation to a level
of about ~.0, base is injected in order to bring the pH
of the system back up to the desired 6.3 level. Ac-
cordingly, the pH of the medium using this technique is
constantly maintained between 6.0 and 6.3, and is never
allowed to decrease to the levels of acidity reached in
traditional processes. rJhile this approach (sometimes
referred to in the art as "externa] pH control") has
achieved substantial usage in the cheese making art, a
number of problems remain. First, whi]e the 6.0-~.3 pH
range is sometimes preferable from the standpoint of
bacterial growth, continual pH maintenance within this
range can upset typical enzymatic systems and, in the
case of Italian cheeses, the coccus/rod ratio ulti-
mately obtained may be adversely affected. ~loreov~r,
in the traditional approach, the drop in pH to the
4.0-5.0 level has the effect to retarding the growth of
pathogens; however, the external pH control system
never permits the pH level to drop to this level, and
accordingly pathogens which would otherwise be in-
activated remain viable in the medium.
Ancther ~ethod develcped in recent years is
described in U.S. Patent ~'o. 4,282,255. This patent
~L2~533~
1 relates to a method for growina acid-producing bacteria
wherein use is made of te~porarily water insoluble
neutralizing agents which are placed directly in the
starter tank. These tablets or bodies include basic
materials, and are designed to slowly and continuously
release base in order to continuously maintain the pH
level, typically between 5 and 7. In practice, and as
disclosed in the referenced patent, these slow-release
bodies require continual stirring or agitation of the
starter tank. Such agitation has been found to precent
problems, inasmuch as it can interfere with proper cell
growth and multiplication. Also, the slow-release pH
control system disclosed in Patent No. 4,282,255 (some-
times referred to in the art as "internal pH control"),
suffers from the fact that the pH level is never al-
lowed to decrease to a point where pathogens are com-
pletely killed.
In ~hort, both the external and internal pH
control systems involve an attempt to control pH within
a re]atively narrow band. Accordingly, a araph of pH
versus time for these processes shows an initial pH
drop to the desired range of pH control, followed by a
generally horizontal (~raphical pattern. In the case of
the external pH control, the graph typically is in the
form of a "sawtooth" by virtue of periodic addition of
base; on the other hand, the internal pH contro] typi-
cally generates a straighter graphical line in this
region, by virtue of the slow, continual re]ease of
base in the starter media and consequent continual
neutralization of acid as produced by the micro-
organisms.
1;215339
1 Summary of the Invention
The present invention provides a greatly
im~roved method of growing acid-rroducing micro-
organisms used in cheese making processes. Broadly
speaking, the process includes the steps of providing a
starter medium in the form of a liquid, which may be
any one of a number of conventional media, including
reconstituted nonfat dry milk solids. The liauid
medium is then inoculated ~ith at least one cheese-
making microorganism, and the medium is allowed to
initially incubate until the pH of the latter drops to
a~out 3.9-5.5, more ~refera~)ly from ahout 4.5-5.3, and
most ~referably below a~out 5Ø The pH of the medium
is thereafter raised, and the system is al]owed to
further incubate until completion.
In one particu]arly preferred form of the
invention, the initial incubation step is carried out
without addition of base and consequent neutralization
of acid produced until the pH is lowered to the desired
point, whereupon the pH is quickly raised at least
about one pH unit and to a ~evel of from about 5.5-7.5,
and more preferably from about 6.3-6.5. Such rela-
tively quick pH raising is most advantageously accomp-
lished by direct addition of liquid base, such as a
base selected from the group consisting of sodium
hydroxide, potassium hydroxide, ammonium hydroxide, and
calcium hydroxide, or any other suitable food qrade
base. Moreover, such quick pH elevation shou]d he
accomplished within a period of up to ahout
3 minutes.
Typically, the initial ~H of the inoculated
medium at the outset of the initial inoculation is
within the range of 6.0-7.5, and the medium is normallv
maintained at a temperature of from about ~0 to 115
degrees Fahrenheit during the initial and further incu-
~l2~S339
l bation steps. As noted, the culture medium can be any
one of a number of heretofore known media, but broadly
should be in the form of an aqueous composition having
milk-derived nutrients dispersed therein. As noted
hereinafter~, a particularly preferred medium includes
whey and a minor amount of lecithin therein, and this
medium has been shown to aive advantageous results when
used in conjunction with the methods hereof.
At the ccnclusion of the preferred procedure,
the titratable acidity of the medium should be from
about 0.5-1.8, and preferably from about 1.0-1.5. At
this point the medium is cooled in order to ter~inate
the incubation, typically to a temperature of from
about 35-60 degrees Fahrenheit.
While the method hereof can be usèd to good
utility in connection with a wide variety of cheese-
making microorganisms, it is particul~rly preferred in
con~unction with cultures used for the manufacture of
Italian cheese such as mozzare11a. In such case, the
medium is inoculated with a mixture of coccus and rod
microorganisms, and the overall process is carried out
so that the final coccus to rod radio is from about 2:1
to 5:1.
As noted ahove, a serious practica1 problem
encountered in connection with so-called internal pH
systems stems from the need to continuously stir or
agitate the starter tank during the incubation. It has
been found, however, that the system of the present
invention need not include such continual agitation,
and indeed it is preferred that the medium be held
essentially quiescent during the initial and further
incubation steps referred to above.
In contrast to the external and internal pH
systems, a graph of pH versus time in connection with
the preferred process of the present invention will,
33g
1 generally speaking, resemhle a "V". The first, down-
wardly extendina les of the "V" depict:s the initial
drop in pH, whereas the second, upwardly extending leg
of the "V" represerts the preferred, relatively sharp
rise in pH ~lhich occurs upon the addition of base.
Durin~ the incubaticn after the pH level has
been elevate~, the FH again beains to drop because of
acid production. Although it is within the ambit of
the invention to again add base to elevate the pH when
it drops to, e.a., below 5.0, it has ~een found that
such additional pH elevations do not give any material
advanta~es. ~ccordingly, it is preferred to have but a
single pH elevation during incubation.
Description of the Preferred Em~odiment
. . . _
The following examples illustrate the methods
of the invention, and compare the same to prior methods
in order to demonstrate the superiority of the pH
modification hereof. It is to be understood, however,
that the examples are for illustration purposes only
inso~ar as they describe pH modification methods in
accordance with the invention; therefore, nothing in
these illustrative examples should be taken as a limi-
tation upon the overall scope of the invention.
EXAMPLE 1
In this series of tests the efficacy of the
method of the invention were tested as compared with
the traditional method of microorganism arowth, usin~ a
total of four separate starter media.
The media selected for this series of tests
included: (1) nonfat dry milk solids dispersed in
water to a 12% solids level; (2) a commcrcial~y
availa~le pha~e-resistant initially dry medium sold
under the designation "Thermostar" hy Marshall ~ivision
~21S33~
1 of the Miles Laboratories, and reconsituted in water to
an 11% solids level; a commercially availa~le pha~e-
resistant, initially dry medium sold under the designa-
tion "Actilac" by Galoway West & Company of Fondulac,
Wisconsin, and reconstituted to a 11% solids level; and
preferred test medium containina lecithin used at a,7%
solids level.
The most preferred lecithin-containing start-
er media is initially in the form of a dri,ed compc-
sition which is then added to an aqueous system to give
a reconstituted liquid starter medium. This compo-
sition includes the fol].owing components:
1;~1533~
TAsLE I
Parts hy Wt. of Dried
InitialComposition
InaredientQuantityl(Dry Basis)
Stimula~t 700 lbs. 4.120
Nor.fat dry milk
solids 900 lks. 5.290
Sodium tetra
phosphate80n lbs. - 4.700
Disodium
phosphate550 lbs. 3.230
Monosodium phos-
phate 800 lhs. 4.700
Man~anese
chloride 400 gr. 0.005
Ferrous ammonium
sulfat~ 40C gr. O.OQ5
Lecithin 5 ~allons 0.290
Sweet 3iquid280,000 lbs.77~660
whey
Total weight or quantity, including free water, of
starting in~redients
Lecithin in liquid form, 50~ by wt. solids; or could
be in the form of a dried powder
3~ay alternatively be derived ~y mixin~ 13,198 ]bs of
dried whey with 266,802 lbs. of water
The preferred dried powder media composition
is made as follows. In the first step, 700 pounds of
the stimu'ant (a dried mixture of corn steep solids and
sweet whey solids described in detail below), alon~
with 900 pounds of the nonfat dry milk solids are mi~ed
with the 280,QOQ pounds of sweet whev (either raw or
pasteurized, normally pasteurized). After sufficient
mixing to disperse the ctimulant and milk solids, the
mixture is neutralized by the addition of sodium hy-
droxide (5Q~) to a pH of 7. The pH-adjusted mixture is
then thermally evaporated under vacuum conditions to a
~5~39
- 10 -
1 41 percent solids level, whereupon the mixture is
cooled to 50 degrees Fahrenheit and transferred to a
final mixinq tank.
A phosp}late/minerals/lecithin premix is
prepared separately from the mixture of stimulant,
dried milk and whey. This premix is made ~y adding 375
gallons of water at 9~ degrees Fahrenheit to a l,OOn
gallon mixing tank. Next, 8~0 pounds of sodium tetra
phosphate is added, fcllcwed ~y 800 pounds of mono-
sodium phosphate and 550 pounds of disodium phosphate,
all with constant agitation. The next step involves
dissolving the 400 grams of ferrous ammonium sulfate
and 400 grams of manganese chloride in a small amour.t
of water, whereupon these minerals are added to the
agitated mixture of water and phosphates. The 5 gal-
lons of lecithin is then added to the premix tank,
again with sufficient agitation to ensure homogeneity.
This premix is then added to the mixture of whey,
stimulant and dried milk solids, whereupon the overall
mixture is agitated overnight and s~ray dried to ahout
4% moisture to yield a flowable, dried, powder-like
material.
The stimulant referred to ahove is made by
taking 280,000 pounds of separated raw whey from the
cheese-making vat (such amount of whey ~eing a separate
quantity from that used in the starter media per se
listed in Table I), and adjusting the pH thereof to a
level of about 8.0 with sodium hydroxide. The pH-
adjusted whey is then evapcrated to a 3~ percent solids
level, and cooled to 50 degrees ~ahrenheit. The eva-
porated whey is then pum~d into a tank containing
42,880 pounds of commercialy purchased corn steep
liquor having a pH of 4.15. Such liquor is obtained
from The Staley Corporation of ~ecatur, Illinois, and
has a 50 percent solids ]evel. This creates a mixture
~S339
1 containin~ about 60 percent b~ weight corn steep solids
and 40 percent hy weiaht whey solids. The 60 percent-
40% mixture is then agitated overnight, filtered and
spray dried to about 4 percent moisture. The res~]tant
dried product is stored in 50 pound bags for subsequent
use in the starter media.
~11 eight of the media samples (two per
media) were heated to 190 degrees Fahrenheit and main-
tained at that temperature for 1 hour, followed hy
lQ cooling to ln2 degrees Fahrenheit. The media wer~ then
inoculated (1~) with ~ stan~ard coccus and rod culture
(Streptococcus thermophillls and Lactobacillus bulgaris)
and incuhated at 102 deqrees Fahrenheit until the pH
carne down to about a.8 (ty~ically 5~7 hours). At tllat
time, one sample of each media was quick]y neutralized
with food c;rade sterile 50 percent sodium hydroxide to
raise the ~H thereof to 6.3-6.5. The respective incu-
bations were then allowed to continue unti~ all titrat-
ab]e acidities were greater than 1Ø In the case of
the "Thermostar" media, the final titratable acidity
level was 1.4, in accordance with the manufacturer's
recommendations. After the appropriate titratable
acidity levels had been reached, the incubations were
terminated hy coo]ing to 40 degrees Fahrenheit.
The cultures grown in the respective media
were tested for pH, titratable acidity, tctal bacterial
count, coccus/rod ratio, and activity, using conven-
tional testing techniques. The results of these tests
are set forth below in Table II wherein those media
subjected to the described pH modification in accor-
dance with the invention are referred to as "neutra-
lized", and those allowed to incubate without rH modi-
fication are referred to as "control~"
33~
TABLE II
Final Tot 3 1
Final Titratable Coccus and Bacterial
Media p~l Acidity Rod Ratio Count Activity
Non-fat
Dry Milk 7
(Control) 4 20 1.02 4:1 140 x 10 0.70
Non-fat
~ry ~ilk
(Neutra-
lized) 4.25 1.09 3:1 220 x 107 0.84
Thermostar 7
(Control) 4.25 1.40 1 1 100 x 10 0.60
Thermostar
(Neutra- 7
lized) 4.23 1.50 1:1 150 x 10 0.69
Actilac 7
(Control) 4.15 1.08 1:1 160 x 10 0.73
Actilac
(Neutra- 7
lized) 4.12 1.04 1:1 250 x 10 0.82
Lecithin
Medium . 7
(Control) 4.35 1.02 4-1 130 x 10 0.72
Lecithin
Medium
(Neutra- 7
lized) 4.32 1.05 3:1 190 x 10 0.83
The foregoing results demonstrate that in
all instances the method of the invention gave superior
results. Bacterial counts were uniformly hi~her, as
were activity readings. Coccus/rod ratios were not
significantly altered as compared with the controls.
L5339
- 13 -
1 EXAMPLE II
In this example the effects of continuous
neutralization and agitation on coccus/rod cultures
were measured and compared with t-he effects of the
methods of the invention.
Nonfat dry milk solids were reconstitute(1 in
water to 12% solids level and four 100 ml. samples
thereof were prepared in respective dilution bottles.
All media samples were then heated to 190 degrees
Fahrenheit and maintained at that temperature ~or 1
hour followed by cooling to 102 degrees Fahrenheit. At
this point the samples were inoculated at 102 degrees
Fahrenheit with the coccus/rod microorganisms decribed
a~ove at a 1 percent leve~ of inoculation.
I'he control media was simply allowed to
inoculate without any pH modification until the titrat-
able acidity level was greater than 1.0~ At this point
the medium was cooled to 50 degrees Fahrenheit.
The external pH control test involved con-
tinual monitoring of thc pH of the medium sample and,
when th~ pH fell to 6.0, it was adiusted upwardly to
6.3 usin~ 50% sodium hydroxide. I'his procedure was
continued for a period of time equal to the incuhation
time of the control, whereupon the medium was cooled to
50 degrees F'ahrenheit.
The agitation test involved continua~ shaking
of the incubated sample, but without any time release
pH modification tablets or t~.e like. Such continuous
a~itation is characteristic of the internal p~ ccntrol
systems described previously.
Finally, the last medium sample was incubated
using the pH modification technique of the invention.
This involved initial incu~ation and monitcring of the
pH of the system until the pH reached 4.~, whereupon 50
33~
l percent sterile sodium hydroxide was added to quickly
elevclte the ~H to a level of 6.3-6.5. The system was
then further incubated without additiona] pH modifica-
tion unti] the titrata~le acidity was greater ~han l.0,
whereupon the medium was cooled to 50 degrees Fahren-
heit.
The results of this test are set forth ir
Table III:
~LA~LE III
Final - Total
~ledia/ Final Titratab]e Coccus/Rod ~acterial
Trea ~nt pH Acidity Ratio Count _ Activity
NFDM/ 7
Control 4.20 l.204:l 140 x lO 0~70
NFDM/
External 7
pH S.45 Q.50lS l 75 x lQ 0.55
NFDM/ 7
Agitation 4.~00.75 7:1 63 x lO 0.51
NF~M/pH
Modifica-
tion of 7
Invention 4.25l.09 3:l. 220 x lO 0.84
~ - - -
The results of Table III clearly demonstrate
the improved results ohtained through use of the method
of the invention. For example, the continuous external
pH control and agitation tests gave low titratahl~
acidities and coccus/rod ratios which were unaccept-
abi.e; in addition, bacteria.l counts an~ activities were
significantly reduced. ~n the other ~and, the method
of the invention gave much improved results as com~ared
with the external pH control and agitation svstems, and
also as compared to the traditior,al incu~ation method
33~
- 15 -
free of pH modificatior.
The various tests referred to in the fore-
going Examples were performed as ~ollows:
_pl~ Hydrogen ion concentration was determined
using Beckman p~ meter -
Titratable Acidity
9 c~rams of medium sample was thoreu~hly mixed
and titrated with 0.1 N sodium hydroxide
using phencphthalein as an indicator. A
faint pin~. color indicated the end point.
Coccus and Rod P~atio
~ one in ten dilution of culture in water was
smeared on a clean glass slide, stained with
methylene b]ue and examined under a com~ound
microscope. The ratio was determined on the
basis of c]ump and indi~idual counts.
Tota] Bacterial Count
The culturec' samples were serially c7iluted insterile phosphate buEfered water according to
the procedures outlined in the Standard
Methods for the examination of dairy products
and plated usinc3 tryptic soy agar ~ortified
with 0.5% yeast extract. The plates were
incubated at 37 degrees Centigrade for 4
days. The counting and expression cf the
test results were done accordinc; to the
Standard Procedures.
S33~
- 16 -
1 ~ctivity Test
2 qrams oE culture was inoculated into 100
ml. of sterile 10.0 g. reconstituted nonfat
dry milk. The nonfat dry milk was pretested
for the inhibitory compounds. The inoculated
milk was incubated at 36 degrees Centigrade
for 45 minutes. At the end of incubation,
the temperature was gradually increased to ~6
de~rees C~ntigrade within a s~an of 30 min-
utes and it was thereafter maintained at that
temperature for a period of 1 hour. The
samples were then chill~d to prevent any
further acid development. Ten grams of the
sample was carefully weighed into a 25 m].
beaker. Ten drops of indicator (Phenoph-
thalein) was added and the entire contents
were titrated against 0.1 N sodium hydroxide
until a faint pink color persisted for 15
seconds. The results were ex~ressed as
percent titratable acidity.
