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FACTORS INNLJLNCING Tfim AJTIVITK OW JLHEJRATEL

CUTTadn CHnnSz Cdnludds

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ChAlfl‘anS II. I 1.1L 1AM SAP P

AN ABSTRACT
Submitted to the College of Agriculture of Michigan State
University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of
MeSde 0F SCILNCE
Department of Dairy

1960

i "' ./
Approved «_",<34;,{3161’9

 

ABSTRACT

In an effort to simplify the problems of cheese cul-
ture propagation in dairy plants, cultures were dehydrated
by the spray system using regular milk drying equipment.
The influence of the following factors on growth of these
organisms was investigated when used subsequent to dehydra-
tion: (1) drying temperatures, (2) incubation periods of
culture prior to dehydration, (3) addition of gelatin to
culture before dehydration, (h) acidity reduction of cul-
ture before dehydration and (5) storage temperatures and
periods of the dehydrated cultures.

The rate of growth (activity) of the dry cultures was
determined by measuring the develOpment of titratable acid-
ity after 1.0 percent inoculation into sterile skim milk
and incubation at 72° F. for 16 hours.

Outlet drying temperatures of less than 165° F.
usually yielded dry cultures with high initial activity
while outlet temperatures above 200° F. seemed to retard
the activity. Acidity reduction of the liquid culture with
calcium hydroxide or sodium hydroxide before dehydration
caused a decrease in initial activity of the organisms.
Slightly higher initial activity was present in the dry

product from cultures with a 16-hour incubation at 72° F.

as compared to 8, 12 or 2h hours. No advantage was obtain-
ed by the use of gelatin in amounts of 0.05, 0.1, 0.5 and
1.0 percent of the liquid culture before incubation and
drying. Activity of the dehydrated culture decreased with
the increase in storage temperature and length of hold.
Most samples stored at 720 F. and 90° F. showed very little
activity after 1 week but at AOO F. good activity usually
prevailed for 1 week and sometimes for 2 weeks. Samples

held at ~15° F. maintained good activity during a year.

FACTORS INFLUMNCING T55 ACTIVITY 0F.DEHIQRATSJ

COTTAGE CHELSE CULTUMbS

By
Chintas alttIAm SAP?

A THESIS
Submitted to the College of Agriculture of Michigan State
University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE
department of Dairy

1960

tefll

A C K N OW L 13' D {3th 1“;th N T S

The author wishes to express his sincere thanks to
Dr. T. I. Hedrick for his suggestion of the research
problem, his valuable direction during the course of this
study, and for the constructive criticism offered in the
preparation of this manuscript.

Gratitude is also expressed to Dr. L. G. Harmon,

Dr. G. M. Trout and Jr. J. R. Brunner for their sugges-
tions throughout the course of this study and to Professor
‘T. S. Lucas and Dr. C. M. Stine for reviewing the
manuscript.

Sincere thanks are expressed to the Dairy Industry
Supplies Association for the fellowship which made this
project possible.

Final acknowledgement is made to my wife, Lorraine,
for her ever ready advice and encouragement, and for the

typing of this thesis.

TABLE OF CJanNTS

I‘LV'II‘HUDJCTIOLJ 000......OOOOOOOOOOOOOOOOOO00.00.00...

UITEi{ATURE BE‘JIE‘U. O...OOOIOOOOOOOOOOOOOOO0.0.0.0...

b4<P1$EiIiliflil§T£XLPROCEDJRL‘JI 0.00......OCOOOOOCOOOOOOCOOO

Culture

Procurement and Preparation ......

Spray Drying Equipment ...................

fiBdUCtion 0f ACidity 0.0000000000000000...

Incubatinl Time 0.0000000000000000.0.0....

Addition Of Gelatin COCOOOOOOOOOOOOOOOOOOO

Storage

Period and Temperature ...........

ACtiVity TeSt 0.0000000000000000.ooooooooo

RESJLTS 00......0.0.0.0.....OOOOOOOOOOOOOOOOOOOOOOO

drying Temperature .......................

i'iedUCtion Of ACidity OCOOOOOOOOOOCOOOOOOOO

Incubation Time ..........................

Addition Of Gelatin OOOOOOOOOOOOOOOOOOOOOO

Storage

JID‘SLJSSIc-J‘N .9...

Period and Temperature ...........

v y - -‘ |j’ ’ :‘1‘1', lf‘ 7'.
SUJLflL’Xf‘i (tail) CJNUUUQIV‘H oooooooooooooooooooooooooooo

LITERATJRS CITED

m
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10
ll
11
12
12
12

INTRJDUCTIUN

Successful transfer and effective prepagation of
lactic acid culture organisms has long been a problem of
the commercial dairy plant operation. Technical knowledge
and approved technique are a necessity if quality starters
are maintained.

Poor quality cottage and cheddar cheese often can be
traced to the inadequate handling of the starter cultures.
Variations from day to day may occur with variances in the
cultures themselves.

The fact has been known for some time that organisms
remain viable in the dry state. Lyophilization has been
demonstrated as an effective means of dehydration, but the
nature of the technique discourages commercial preparation
for direct inoculation into the cheese vats. Spray drying,
a common method for dehydrating whole and skim milk, has
had little use in the desiccation of bacterial cultures.
Since this technique possesses the distinct advantage of
economy over the other methods, this study was initiated
to determine the activity of bacterial cultures surviving
the heat treatment during dehydration, to determine
desirable techniques in the drying operation itself, and
to assure maximum survival of active organisms'in the

dehydrated culture medium.

LITERATURE REVIEW

Christian (1) stated that bacterial cultures have
been known to remain viable in a dehydrated medium. 0r-
ganisms have been found in a dry condition in nature that
were capable of growth. desiccation methods have been
developed for the preservation of stock cultures. The
presence, although undesirable, of viable bacteria in low
moisture dry milk solids, has been known for many years.

Poor and Fithatrick (19) reported that pathogens
could survive in a dry condition. In their experiments
the pathogenic organisms which were subjected to diffused
light, desiccating conditions, dirt and other bacteria
were greatly reduced in numbers. However, Streptococci
remained viable for a period of not longer than 1 week and
Diphtheria bacilli (Corynebacterium diphtheriae) survived
for 3 weeks.

A simple method of dehydration, designed for the
study and maintenance of a stock supply of cultures,
consisted of placing the culture medium in a desiccator
over a dehydrating agent such as sulphuric acid, calcium
chloride or phosphorus pentoxide. Brown (3), Stamp (23)
and Stark and Harrington (2h) reported successful results
using this method.

Stark and Herrington (2h) stated that approximately

 

66 percent of the Streptococci originally present were
able to grow in a culture medium after 97 days under dry
conditions. Less resistant organisms were unable to grow
after 2 or 3 days.

Stamp (23) suspended bacterial cells in melted nutri-
ent gelatin containing ascorbic acid. The samples were
dried in small quantities using phosphorus pentoxide with
pressures of 100 to 300 mm. of mercury and were stored in
vacuo with phosphorus pentoxide at room temperature.

Another method of dehydration consisted of freezing a
small amount of culture by immersing it in a mixture of
ice and salt or dry ice and glycerol. Rogers (21) found
that 0.1 percent inoculation of lactic acid cultures that
were dried by this method was sufficiently active to coag-
ulate milk in 17 hours of incubation at 300 C. Morton
g3 g1. (15) and Swift (26) also reported favorable results
using the same method of drying cultures.

Hammer (9) in 1911 used a variation of the freeze-dry
method to preserve bacteria. He placed strips of filter
paper impregnated with bacteria into test tubes. The test
tube contents were then frozen in an ice-salt mixture and
placed in a desiccator containing sulphuric acid. A
partial vacuum was drawn on the desiccator. The organisms
remained viable for 57 days.

One thousand five hundred different strains of bacte-

ria were dispersed in cotton plugged test tubes of nutri-

ent broth. The samples were frozen by immersion in a
broth of glycerol with solid carbon dioxide within a
desiccator. The samples were re-dried under vacuum with
phosphorus pentoxide for 7 days at room temperature.
Afterwards, the tubes were sealed.

Elser g; 3;. (o) and Flosdorf 33 31. (7) used the
same principle. They enlarged upon the technique used to
develop the "LyOphile" process. In the lyophilization
process the suspension to be desiccated was placed into
containers and frozen by immersion in a dry ice and glyc-
erol mixture. When thoroughly frozen, the samples were
attached to a manifold connected to a vacuum pump. The
moisture was drawn out of the frozen state by means of a
vacuum.

Appleman and Sears (2) preserved legume nodule bacte-
ria by the 1y0philization process. These bacteria remain-
ed viable for periods of 3.5 to h.0 years. Their ability
to grow and fix nitrogen was undiminished as compared to
the corresponding stock cultures that had been prepagated
normally.

Maximum survival of Serratia marcescens was obtained
by Naylor and Smith (16) using the lyophilization process.
Survivals of 100 percent and counts ranging up to S x 1012
per gram of dried material were obtained.

Elser t a1. (6) using their lyophilization method of

dehydration found that such highly sensitive microorganisms

as Gonococcus and Meningococcus could be preserved for
periods up to 13 years when the product was stored in
vacuo.

Rogers (21) obtained varied preservation success by
means of a warm air current passing through the culture
medium. Loss of viable bacteria was greatly reduced when
the water was removed rapidly by a spraying method.

Rogers also reported, "The usual process of drying cul-
tures consists in reducing the percentage of water by add-
ing a dry substance, such as powdered lactose and expos-
ing the mixture to a current of air at a temperature
sufficiently high to cause a rapid evaporation."

Numerous references in literature reported the surviv-
al of microorganisms in skim milk powder (nonfat dry milk).
Downs (5) found a wide variation in the number of bacteria
per gram in dry milk processed by different methods, as
well as in different samples from the same method.

Average bacteria counts in spray process dry milk were
higher than in roller process dry milk. Samples that were
dried from condensed milk had a lower bacteria count than
samples from the same supply that was not condensed.

Supplee and Ashbaugh (25) in 1922 assumed that viable
bacteria counts in excess of 1,000 per gram were caused by
contamination after the powder was removed from the drying
cylinders.

Hunwicke and Jephcott (12) reported that non-spore

forming bacteria were completely destroyed by roller
process milk drying.

Wide ranges in original counts on spray process dry
milk were noted by Macy (13). A decrease in counts was
noted during the first year of storage. Macy also
observed the decrease in organisms was less in samples
from the roller process than from the spray process. He
believed the reason was that the spore forming types which
survived the roller process were able to withstand the
adverse conditions during storage of the dry milk.

Higbinbottom (10) reported a higher survival when dry
milks were spray dried as compared to roller dried. High-
er counts in spray process powders have been reported by
Nichols (17) also.

Roller process powder showed a predominance of pep-
tonizing organisms while spray process powder showed a
predominance of acid producing bacteria according to the
results published by Macy (13). He used the standard
plate count. A predominaice of small sub-surface colonies
resembling Streptococcus lactis were noted.

Kattick at El- (1h) examined the viable microflora of
dry milk and found Streptococci, micrococci and spore
bearing organisms.

Stark and Harrington (2h) tested seven different
species in the dry condition and reported that Streptococci

paracitrovords and Streptococci lactis were the most

viable, while Lactobacillus acidophilus was the least

 

viable.

A definite loss of viability occurred when organisms
were subjected to the dehydration process. Rogers (21)
believed that this might be due to large salt concentra-
tions which occurred with the removal of the moisture.
Anderson (1) stated that organisms undergoing dehydration
were flattened due to the surface tension exerted when the
moisture was removed.

Several factors such as relative humidity, moisture
content, oxygen content, pressure and temperature have
been reported to be involved in the survival and activity
of organisms during storage. Higginbottom (11) reported
that the curve of survival during storage indicated an
increasing rate of survival from 30 to 10 percent relative
humidity followed by a decreased rate below 10 percent.
Proom and Hemmens (20) and Rogers (21) observed that the
loss of viability was much more rapid when the moisture
content of dry milk was high. Rogers (21) and Stark and
Harrington (2h) reported that oxygen exerted a deleterious
effect on bacteria in dry milk during storage.

Naylor and Smith (16) and Rogers (21) observed that
vacuum.packed dehydrated culture medium preserved viability
more than nitrogen or hydrogen packed samples. Christian
(A) reported a correlation existed between the pressure

under which a culture was stored and the percent viability

8
of lyophilized cultures. Eight hundred thirty-five microns
f mercury was found to be the highest pressure under which

MlCPOCOCCJS pvogenes variety aureus could be stored.

 

Rogers (21) observed that at a temperature of 00 C.
the loss of activity was slow but at 300 to 370 C. the
loss of activity of Streptococcus lactis was rapid. in»

Samova-Kaukova (3) noticed that the 0.15 to 3.0
percent filtrate of 11;”id cultures of Uidium lactis

(Geotrichum candidum) caused increased growth of dehydrat-

 

ed Streptococcus lactis organisms when they were grown on
an agar medium. Speck and Myers (22) reported that dried
skim milk cultures of Lactobacillus bulgaricus did not
develop many colonies when reconstituted at 210 to 250 C.
A much larger number of organisms grew when the samples
were reconstituted at 370 to 50° 0.

Rogers (21) reported that the total number of bacteria
in a milk culture may be increased by adding dibasic
potassium phosphate. The organisms in the dry product
processed from this culture were less viable than those
similarly treated except that the culture medium was not
neutralized. When calcium carbonate was used to neutral-
ize the culture before drying, the viability of the organ-

isms in the dehydrated product was not increased.

EXPmRIMENTAL PROCEDURE

Culture Procurement and Preparation

Several cottage cheese cultures were obtained from
culture supply companies for use in this investigation.
One of the cultures was selected for the experimental work
and transferred into eight duplicate samples. The active
cultures were propagated daily, with the exception of
Sundays, according to the accepted procedure.

Raw skim milk was obtained from the Michigan State
University Dairy Plant. Ten gallons of milk were heated to
1900 F. in a waterbath and held at that temperature for 30
minutes in a stainless steel can. At the end of the 30-
minute heating period, the skim milk was cooled to 720 F.
with flowing water. Inoculation was then made with one
percent of the selected culture. The starter was held 16
hours at 720 F. and then cooled to 52° FR with water.

Next it was removed and placed in a cooler at hOO F. until
dehydration.

Titratable acidity determinations were made on the
culture by using 0.1 normal sodium hydroxide and five drOps
of a one percent alcoholic solution of phenolphthalein
indicator in a Q-gram sample.

Immediately before dryi.g, each sample was homogen-

ized with a Cherry-Barrell "200" series single stage

10
homogenizer at 1,500 psi. Following homogenization the
sample was placed in the pressure container to which the
feed line to the nozzle was attached. after the drier
temperature was allowed to reach equilibrium the drying of
the culture was begun. Powder was collected in a 2-quart

glass container at the base of the cyclone collector.

Spray Drying equipment

A laboratory size Swenson spray drier was used in
this investigation. The drier consisted of a direct flame
gas heater, drying chamber, cyclone collector, exhaust fan
and compressed air atomizing system. The gas heater used
to heat the filtered air was controlled by a solenoid
valve. The drying chamber was constructed of stainless
steel consisting of a vertical cylindrical section 60
inches high with a 37-inch inside diameter and a conical
bottom. The base of the cone was the product outlet; the
drying air inlet was located in the conical section. A
water cooled atomizing nozzle was used. It was connected
to the pressure feed tank. A pressure regulator maintain-
ed constant pressure. Instrumentation of the drier includ-
ed an atomizing air pressure gauge, feed pressure gauge,
outlet air indicating thermometer and an inlet air temper-
ature thermometer and controller.

Capacity was approximately 30 pounds of water removal

per hour from a feed solution at 1300 F3 when Operated with

11
an air inlet temperature of 3500 F.

Inlet air and outlet air drying temperatures were
recorded for each trial from the inlet temperature control
dial and outlet indicating thermometer. Accuracy of the
indicating thermometer was checked periodically. A range
in inlet air temperature was obtained by adjusting the
temperature setting which regulated the solenoid valve on
the gas line. The outlet air temperature was varied by
regulating the inlet temperature and the amount of product

fed into the drying chamber.

Reduction of Acidity
The acid in the culture was reduced with sodium

hydroxide and calcium hydroxide. A control sample was
desiccated in each series. Subsequent samples in each
series were reduced in acidity to 3.60, J.h0, 0.20 and

0.0 expressed as percent of lactic acid. The neutralizing
agent was mixed with sterile, distilled water in the ratio
1:10 and slowly poured into the cultured skim milk with

continuous agitation by means of a stirring rod.

Incubation Time
Samples in the trials involving four incubation times
were held constant at 720 F., after an inoculation of 1.0
percent. These samples were removed from the incubator at

3, 12, 15 and 2h hours, respectively, and dehydrated

 

 

12

. 0
immediately after cooling to 52 F.

Addition of Gelatin
Gelatin was addei to a series of samples. They were
prepared the same as described previously with the excep-
tion that the weighed amount of gelatin was dissolved in
600 ml. of hot water and was added to the skim milk before
pasteurization. additions were 0.05, 0.1, 0.3 and 1.0

percent calculated on the basis of the total milk solids.

Storage Period and Temperature

H
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(‘f

ely after desiccation the dry cultures were
placed in small amber bottles with screw caps. These were
labeled and stored at Ado, 72° and 90° F. A sample at
each storage temperature was tested for activity after
:ach 7-day period for a weeks or until the activity was

25 or less. Samples were placed in storage at -150 F.

and tested for activity after 1 year.

Activity Test
Preliminary trials indicated that the standard plate
count was not a good index of the rate of growth of the
organism from a dry culture. The best indication was the

ability of the organisms to deveIOp acidity in skim milk.

The test called "Activity Test" consisted of the following:

(1) 99 ml. of skim milk was placed in 180 ml. (6 ounce)

13
medical vials with caps; (2) these vials were heated to
14900 F. for one-half hour in an autoclave; (3) samples
were allowed to cool to 1600 F. and then were cooled with
flowing water to 720 F.; (A) 1 gram of dry culture was
weighed and transferred to the medical vial with the skim
milk; (5) the mixture was shaken until the dry culture was
uniformly dispersed; (6) the inoculated samples were incu-
bated for 16 hours then promptly cooled in flowing cold
water; (7) a 9-gram sample was weighed into a titrating
dish and the percent acidity determined by using 0.1 N.
sodium hydroxide with five drops of phenolphthalein as the
indicator, and (3) the results were expressed as activity

which represented the percent acidity multiplied by 100.

yr; “ =21

w—— —.

i

 

RLSJLTS

Drying Temperature

Data of Table I show the relationship of outlet air
drying temperature to initial activity of the dehydrated
cultures. Observation of the data of Samples 9, 10, 13
and 1h indicates that the outlet rather than the inlet air
temperature was critical for optimum preservation of activ-
ity. Inlet air temperature for these four dryings was
5600 F. Sample 9 had an outlet temperature of 1500 to
165° F. with an initial activity of 83. Outlet air temper-
ature for Sample 10 was 1600 to 1650 F. with an activity of
30; Sample 13 with 1530 to 1630 F. and activity of 3a and
Sample 1h with ISBO to 150° F. and activity of 83 were
within a close range for temperature and activity.

Samples 1, 2, u and 7 also had an inlet air tempera-
ture of 5600 F. A low initial activity of 22 was observed
in Sample 1. It had an outlet air temperature of 2000 to
2050 F., approximately MOO higher in temperature than
Samples 9, 10, 13 and 1h. Sample 2 had an activity of 25
with an outlet air temperature of 1900 to 2030 F. Sample
h with a 100 lower outlet air temperature than Sample 2,
had activity of 63. Sample 7 with an outlet air tempera-

ture of 1730 to 1770 P3 had an activity of GA.

In reviewing the difference in activity from various

 

 

 

nary.
.; -‘

TABLE I -- The inflge

 

initial activity

 

n
2.12

as BE 0
the 'e

15

utlet drying temperature pg

v.“ —

dflhydrateg lactig culture

 

Inlet air

Outlet air

Acidity before

Initial—

Sample_(degrees_F.) (degrees F.)Jdrying (percent) activity

 

1

<3 (3 -q 0\ U1 #7 kn to

H F‘
ea <3

5600
560
520
560
520
1180
560
520
560
560
the
Bio
560
560
uso
too
t30
520
360
320

360
290

200-205
190-200
190-195
185-190
173-182
175-180
173-17?
165-170
160-165
160-165
160-165
150-165
153-163
153-160
153-15?
153-157
its-150
ltS-ISS
IMO-1&5
130-135
128-132
120-125
120-125

0 0.80

.81

.80

.30

.80
.80

22
25
is
65
AS
70
61+
SS
83
80
81
76
8h
33
S7
85
82
78
87
86

'7‘

-.—- .s ‘._-.— _-

f‘

16

outlet air temperatures, one observes that a general

increase in activity corresponded to a decrease in outlet

air temperature. A low activity of 22 for Sample 1 was

recorded for the highest outlet air temperature (2000 to

2050 F.). Sample 19 had an outlet air temperature of 11100

to 11150 F. and the highest activity of 37. Two exceptions 3*“

were noted. The activity was 57 in Sample 15 and in Sample

-fll: ‘n-un

22 it was 56 although both had outlet air temperatures '",
below 1600 F. The relationship of outlet air temperature
to initial activity is shown graphically in Figure l.

The principal purpose of Table 2 is to show the effect
of acid reduction on activity. However, the data in Table
2 confirmed the fact that activity increased with a de-
crease in outlet air temperature. Sample 2h with the
highest outlet air temperature (2000 to 2050 F.) had an
initial activity of do compared to Sample 29, which had an
outlet air drying temperature of 1600 to 1650 F. and an
initial activity of 80, or Sample 33 with an outlet tem-
perature of 11150 to 1500 F. and an 83 for the initial
activity. The same trend applied to the results of the

other samples recorded for Table 2.

Reduction of Acidity

 

The effect of the reduction of culture acidity before
drying on the initial activity of the dried culture was

studied. Data are presented in Tables 2 and 3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Outlet Air Temperatures
F's l. ”he effect of outlet air drying temperature

- as.

on initial activity of dehydrated lactic cultures.

Jot ~.‘a.a¢‘a.-l 5.1.!

18

Samples 27, 36, Q2 and Ah were reduced in acidity to
four different percentages with sodium hydroxide. They had
similar drying conditions. Sample 27 with an acidity
reduction from 0.33 to 0.60 percent had an initial activity
of 71. Sample 36 with the acidity reduced to 0.h0 percent
had an initial activity of h5. Sample h2 was reduced to
0.20 percent and the initial activity was to. Sample Ah
was neutralized to the phenolphthalein end point. The
resulting initial activity was 20.

The relationship of the amount of acidity reduction
in liquid cultures to the decrease in activity of the
corresponding dried culture is presented graphically in
Figure 2. Activity is plotted against outlet air tempera-
ture with symbols for the designation of the final percent-
age of acidity. The highest activity present on Figure 2
is a control sample. The lowest activity occurred in the
samples that were neutralized to the phenolphthalein end
point before drying the culture.

The data obtained on the activity of cultures that
were reduced in acidity with calcium hydroxide prior to
dehydration are presented in Table 3. Low initial activity
was observed in 11 of the 1h samples which were reduced in
acidity to 0.60, 0.h0 or 0.20 percent. Three samples

were exceptions to the general trend since these activities

were above 25.

 

9...... ‘ ‘7‘?” ‘7
.
’1

19
T3855 2 -- T‘ne influence Lf aciditv reduction Lf
ch=ese culture witi souium Lydroxide Ln the initial

activity Lf the dried culture

 

 

 

 

 

Inlet air Outlet air Acidity before Tnitial
Sample (degrees F.) (degrees F.) drying (percent) activity

 

2h 5600 200-2050 0.60 no
25 560 105-205 .60 51
26 520 183-192 .60 54
27 520 178-182 .60 71
28 560 163-167 .60 70
29 520 160-165 .60 80
30 560 158-162 .60 71
31 085 153-157 .60 78
32 500 150-155 .60 71
33 660 105-150 .60 83
36 560 200-205 .60 36
35 520 188-192 .60 30
36 520 176-182 .60 65
37 520 160-165 .uo 43
38 520 155-160 .60 55
39 5.20 1240-1115 .110 70
60 560 200-210 .20 26
01 520 190—195 .20 28
62 520 180-185 .20 no
63 520 168-172 .20 01
in 560 178-182 .00 26
05 520 160-165 .00 20

16 520 155-160 .00 25

“"T

‘lfl—ga‘j
‘

 

20

T5805 3 -- The influence 2; acidity reduction 9; cheese
culture with calcium hydroxide 9g the initial activity
gf the dried culture

 

 

 

 

 

 

 

Inletfiair Outlet air Acidity 665353 IEYEIEI‘

Sample (degrees F.) (degrees F.) drying (percent) activity
67 560° 175-1800 0.60 23
08 520 160-165 .60 21
M9 520 153-157 .60 53
50 520 100-105 .60 22
51 520 185-100 .60 20
S2 520 175-185 .MO 22
S3 520 160-165 .00 20
SM 520 153-157 .60 25
SS 560 200-205 .20 20
56 520 190-195 .20 21
S7 520 180-185 .20 22
58 520 165-170 .20 23
S9 520 150-155 .20 58

60 520 105-150 .20 59

‘C v -- -.. A -‘-.-_..---..-'---

 

 

 

L-v- _ -.-‘n_ul1

21

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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O O O O O O
O O O O O 0
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Outlet Air Temperature
c>-— Control
Ԥ._.Acidity reduced to 0.60 percent

||-- Acidity reduced to 0.h0 percent

)(-- Acidity reduced to 0.20 percent
C3-- Acidity reduced to 0.00 percent

Fig. 2. Activity of cultures with acidity reduced to
0.60, 0.60, 0.20 and 0.00 percent and dried at various

outlet air temperatures.

 

 

 

22
Incubation Times
Initial activities of the dried lactic culture in
relation to the incubation time of the culture before dry-
ing are shown in the data of Table A. hesults of the
trials seemed to indicate that incubation of the culture
for 8 hours caused a lower initial activity as compared to p

l2, l6 and 2h hours. The minimum initial activity observed

a . wt...“

was 26 for Sample 89 (3 hours of incubation). There ’ (a
appeared to be a larger difference of activity among the

samples with 3 hours incubation than among the samples

with l2, 16 or 2h hours of incubation. The difference was

3 or less among the samples having l2, l6 and 20 hours of

incubation.

In the first series (Samples 89 to 92) an activity
difference of 3 was observed between the sample with 8
hours incubation and the sample with 12 hours, with a
spread of 3 in activity between l2, l6 and 2h hours
(Samples 90, 91 and 92). In the second series (Samples
93 to 96) the activity was MS for 3 hours, Cl for 12 hours,
Sh for 16 hours and bl for 2h hours. The dry cultures in
the third series had good initial activities, however, the
same trend was apparent. The sample with 3 hours incuba-
tion resulted in an activity of 72 whereas samples with 12-
16- and 2h- hour incubation periods had activities of 80,
81 and 30, respectively. The fourth series of samples had

activities that were slightly higher (7h, 82, 3h and Bl)

23
than the third series for the corresponding incubation

periods.

Addition of Gelatin

Gelatin in the amounts of 0.05, 0.10, 0.50 and 1.00
percent.was added to skim milk before pasteurization,
inoculation, acid development and dehydration. Table 5
presents the results of the activities of these trials.

Both the controls and the samples contailing gelatin
(0.05 percent) had a high initial activity. The activity
of the samples with 0.10 percent gelatin added was compa-
rable to the controls and in the same range of activity as
the samples with 0.50 percent gelatin added which also was
not improved. The lowest activity was 70 and the highest
36 (0.50 percent gelatin) compared to the activity range
of hfl to 87 for the controls. The final series of samples
had 1.00 percent gelatin addition. The activities were
slightly higher than those of the control, but the differ-

ence was considered unimportant.

Storage Period and Temperature
Samples of dehydrated lactic cultures were held at
o 0 o u - , .
hO , 72 and 90 r. and were tested weekly for activ1ty.
4 1 o O
a few dried cultures were stored 1 year at -15 F. and

were tested only at the end of this period.

Of the nine samples held a year at -150 F. (Table 6),

 

 

 

”IJTV
.',|"’_.

 

 

25

~.— ~-

from cultures with di ferent incubation times

 

 

 

Culture incubation Outlet air Moisture Initial

 

Sample (hours) (degrees F.) (percent) activity
89 -8 190-2000 6.6 26
91 16 190-200 8.3 28
92 26 190-200 0.7 31
93 8 l”‘-100 5 6 L5
94 12 175-130 5 8 51
qg 16 175-180 5.6 St
00 3 175-180 5.7 51
97 8 160-165 6.2 72
98 12 ' 160-165 6.0 80
99 16 160-165 6.3 81

100 28 160-165 6.1 80

101 8 155-160 7.7 70

102 12 155-160 7.5 2

103 1( 1 5-160 7.7 80

re
5
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u
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100 81

3 r0

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25

 

a>mbz m :1 ancwmw modH<HMM,mM darkneedea Hmonwo Oswacucw ewes mwwwodona
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'II’.

 

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06Hmeus 08-msee 0606500 11 eewenue 0mmmee .!!:::|1
Apmeoosnv Apmeocndv Amadomsdv Apmwoczdv Aemdoonav
aeHmH Hnwdwmw Hnwdpmw annwmw Hnwnwmw asudwmy
558003 medH<Hd% monw<gww medw<wnw medw<wd< monw<wn%
H 0.00 :0 9.0.0 00 0.0 3 0.0.0 0 H00 :-
N 0.00 N0 0.0m um Oowo Nw 0.m0 00 H.00 mm
0 0.00 0H 0.0m 0m 0.00 00 0.m0 00 0.00 00
r 0.00 mm 0.0m mm 0.H0 am 0.m0 9H H.00 mm
m 0.00 wN 0.0m do 0.H0 am 0.m0 mm H.00 wO
0 0.00 00 0.00 00 0.m0 00

 

26

six had an activity of 70 or above. The activities of the
remaining three were 52, 5h and 53. These activities
indicated that only a small change occurred during storage.
The activity of dehydrated cheese cultures (Table 7)
stored at hOO F. for 7, 1h and 21 days decreased as the
storage period increased. A wide difference in the amount
of decreased activity was noted. The samples with high
initial activity, for example 32 or above, generally de-
creased little in activity during the first 7 days of
storage at hOO F. Most of the samples with initial activ-
ities hetween L0 and 70 decreased substantially in activity
during the first 7 days. The samples with initial activi-

ties below hO did not decrease as much during the same

{-4

perio .

At the end of 1h days storage the activities of the
samples with high initial activity decreased moderately.
These samples had a marked decrease wh n tested at the end
of 21 days. None of the activities were above 63 and many
were below 50. At the end of 23 days these same samples
had less activity. None were above h3 and most were be-
tween 30 and hO in activity.

0f the group with the initial activity between h0 and
70, the activity at the end of 1h days had decreased
generally to within the range of 20 to 35. At the end of

21 days all of this group were below 25 in activity. None

of the remaining samples, those with initial activities

_,“...4I-

27
below hO, had activities above 25 at the end of the 1h-day
storage at hOO F.

The activities dropped rapidly during a 7-day hold at
720 F. The average initial activity of all samples in
Table 7 was 63.2. The average activity of these samples at

the end of 7 days at 720 F. was 2h.l with a range of 16 to

us.

'K‘;- in..- “I. ' ii"
'?

The average activity for the duplicate samples that

 

were held 7 days at 900 F. was slightly lower than those at
720 F. No sample held at 900 F. had an activity above 2h
at the end of 7 days. The average activity of 19 samples
was 20 and the range was 17 to 2h.

The data on Table 8 present the storage results of
the activity test on the samples of dried lactic culture
from cultures reduced in acidity with sodium hydroxide
before drying. Nine of the 10 samples with acid reduced
from 0.32 percent or above to 0.60 percent decreased in
storage at hOO F3 during 7 days. The average of the ini-
tial activities was 66.9 while the activity at the end of
7 days averaged 57.5. After 1h days the average activity
decreased to hh.l. Four of 10 samples were below 25.

The six samples with acidity reduced to 0.h0 percent
before drying had an average initial activity of hB. All
but one had a definite decrease in activity at the end of

days at hOO F. The average was 32.8. Similar results

were obtained on the samples with 0.20 and 0.00 percent

28
level of acidity. The amount of decrease in activity was.
less because the initial activities were low.

All samples with reduced acidity had very low activi-
ties at the end of 7 days when held at 720 or 90° F. Many
cultures held under these conditions had no growth as
measured by the activity test.

The samples of cultures with gelatin added prior to
dehydration and held in storage at hOO, 720 and 900 F. are
reported in Table 9. Samples 61 through 65 with 0.05
percent gelatin added had an average activity of 70.2 after
7 days at MOO F., 61.2 after 1h days and h2.2 after 21
days. At the same storage temperature (11.00 F.), samples
66 through 71 (0.10 percent gelatin) had average activities
of 62.7, 57.2 and 37.8 after 7, IA and 21 days, respective-
ly. Average activities of six (Samples 72 through 77 with
0.50 percent) were 69.5, 57.8 and hh.0 for 7, 1h and 21
days, respectively. The largest addition of gelatin was
1.00 percent in Samples 78 through 82. The average activi-
ties were 7h.8, 61.0 and h3.h for storage periods of 7, 1h
and 21 days at hOO F.

Data on Table 9 also show activity results of the
samples held 7 days at 720 F. The average activities of
the samples with 0.05 percent gelatin was 23.8, with 0.10
percent 2h.2, with 0.50 percent 25.7 and with 1.00 percent
28.0. All 22 samples with the four different amounts of
gelatin added (0.05, 0.10, 0.50 or 1.00 percent) had

29
activities of 21 or 20 indicating a definite decrease

when held 7 days at 900 F.

 

 

 

30

TABLE 6 -- The activity 2f dehydrated lactic cultures
held one year a_t_ -150 E.

 

 

 

 

 

 

 

Sample Initial activity Activity (one year) 7
72 7O 52
7h 78 7;
7E 31 7t
76 32 7o
77 86 76
78 7h 58
80 8: 73
31 35 7h

82 86 5h

 

 

 

31
TABtE 7 -- The influence 2; storage 9n the activity 9;

dehydrated lactic cultures

 

 

 

 

 

 

LOU F. _72° F. 900 F.
Initial Activity of samples after storage (days)
Sample activity 7 lb 21 28 7 7
1 22 20 2o - - 22 2o
2 25 - - - - - -
3 us 36 21 - - 25 20
u 65 59 26 - - 22 21
5 is 31 21 - - 22 -
6 7o 58 37 - - 20 2o
7 6t 89 28 - - 21 22
8 SS 38 29 - - 22 -
9 83 82 75 58 36 A6 21
10 80 79 h9 29 - 2h 19
11 81 76 55 33 - 20 2o
12 76 7t 73 59 Al MB 2A
13 an 83 75 5t 32 25 19
1h 83 82 76 a9 29 25 2o
15 57 A5 3h - - 21 -
16 85 8t 81 63 A3 20 2o
17 32 79 77 us 33 32 19
18 78 71 2 - - 19 2o
19 87 83 81 52 38 2o 19
2o 86 82 75 t1 28 20 i9
21 75 78 (Broken Sample)- 19 17
22. 56 u6 21 - - 16 21

23 82 h} 18 - - 20 19

 

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O ,0,
too a. 72 33.;30 43.-
Activity of samples

 

Acidity Initial after storage (days)
Sample (percent) activity 7 11 21 23 7 7

‘m-

 

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2 20 17 - 20 13

25 .60 51 c9 21 - - 21 13

26 .60 5h 58 36 2; - 20 19

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27 .60 71 61 18 31 18 2o 19
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33 .60 83 30 73 38 21 32 25

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“."-‘_‘“-‘ ‘m-’ -- ’-

faBuE 9 -- The influence of st

 

“orage on the activity

01 dehydrated lactic cultures from trials with variOJS

 

additions of gelatin

 

”n-“ W-..-

-‘

-.

 

 

 

 

and-“ o'- O-

33

 

 

 

W690 2. 372° a. 900 F.
Gelatin Initial :Tfiirigio:§_:a€§:::)
Sample (percent) activity 7 16 21~_§8 _» 7 7 Ag
61 0. 05 76 70 5o 36 21 31 20
62 .05 78 72 65 66 26 28 20
63 .05 82 81 78 56 33 20 2o
66 .05 82 80 76 53 31 20 20
65 .05 79 68 37 22 - 20 20
66 .10 71 68 38 26 - 25 20
67 .10 73 58 37 22 - 26 20
68 .10 81 78 73 51 36 28 21
69 .10 85 80 75 66 39 26 21
70 10 86 86 80 62 63 20 2o
71 .10 86 68 60 22 - 2o 20
72 .50 70 36 32 2o - 23 20
73 .50 80 71 62 69 29 26 20
76 50 3 76 59 68 28 28 20
75 .50 81 78 76 60 63 31 20
76 .50 82 80 69 56 38 25 2o
77 .50 86 78 69 33 21 23 20
78 1.00 76 56 31 20 - 27 20
79 1.30 82 80 71 53 31 29 20
80 1.00 86 30 76 57 36 36 20
81 1.00 85 78 69 51 31 30 2o
82 1.00 86 82 58 36 20 20 20

fl

DISCUSSION

A review of literature indicate that researchers
generally have used growth on standard agar plates as the
measure of survival rate and viability of the desiccated ‘Fj
cultures. In this study this measure did not satisfy all
the requirements necessary for the evaluation of the
ability of dried lactic cultures to develop acidity and
coagulate skim milk when used as an inoculum in a normal
settin time. A test was desirable that would indicate
the ability of dried cultures to give suitable results
for cottage cheese making. Accordingly, the growth of the
organisms (activity) was measured by the titratable
acidity develOped when pasteurized skim milk was inoculat-
ed with dried culture and incubated 16 hours at 720 F.

Greater differentials were noted between inlet and
outlet air drying temperatures than were common in
commercial drying of similar products by the spray process.
This probably can be explained by nature of the drying
chamber of the experimental spray drier. The chamber was
only 60 inches high so the contact time between milk
particles and heated air for dehydration was relatively
short.

Previously stated was the fact that the outlet drying

temperature was more critical to optimum activity than

35
was the inlet temperature. Activity of cultures dehydrat-
ed at the high inlet temperature of 5600 F. may appear
excessive for culture activity and too high to be practi-
cal for a commercial drying temperature. However, evapora-
tion of moisture from the cultured milk particle takes
place at the wet bulb temperature. The reason for the
outlet air temperature being critical to culture activity
probably can be explained by he temperature of evapora-
tion of the culture particle which is influenced by outlet
temperature, especially near the completion of drying.

One should consider the cooling effect of evaporation
of moisture from the culture particle and its relation
with wet bulb temperature. The actual temperature attain-
ed in the particle is considerably lower than the inlet
temperature of the air until evaporation begins to cease.
Other investigators have already shown the critical range
for denaturization of protein material occurs in the range
of 80 to 90 percent solids rather than in the dry state.
This same relationship may be important in an explanation
of culture activity.

Observation of increasing activity with decreasing
outlet air temperatures within the ranges studied can be
explained in view of wet bulb temperature or evaporation
temperature. Lower evaporation temperatures would be
indicative of less heat shock to the organisms during

dehydration and consequently more viability and activity

 

potential in the dry culture.

Less activity was noticed in cultures with acidity
reduced by the use of sodium hydroxide prior to dehydra-
tion. The neutralized dried cultures showed practically
no activity. Activity decreases were progressive in
samples as the acidity was reduced to 0.60, 0.60, 0.20
and 0.0 percent. Acidity reduction with calcium hydrox-
ide also caused initial activities to be low, in fact lower
than the samples comparably treated with sodium hydroxide.

In the investigation of the nature of the effect of
acidity reduction before dehydration on activity of dried
lactic culture, a few additional trials were conducted.
Cultures were removed after 8 hours incubation. The
titratable acidity was 0.50 to 0.52 percent. Half of the
sanples were acidified with lactic acid to 0.83 percent
since this was the approximate average acidity of the
cultures and usually resulted in good activity in the dry
state.

The average initial activity of the cultures dehydrat-
ed without acid added was 76 and of the acidified cultures,
79. The titratable acidity due to the higher acid content
(acidified culture) of the 1 percent inoculation would
increase the activity approximately 3.0. Subtraction of 3
from 79 leaves 76. Consequently, any protective effect of
lactic acid in the maintenance of culture viability during

drying would aopear to be negligible on the basis of these

r.‘ 1:: :
‘ '2-

g .

 

 

 

 

 

37
data.

An improbable factor for less activity of samples
reduced in acidity could have been the demise of the organ-
isms caused by the acid reducing agent (sodium or calcium
hydroxide) at the time the acidity was reduced. This did
not seem a feasible solution when bacteria counts before
and after acid reduction were compared. The standard
plate counts remained practically unchanged.

The greater salt concentration resulting from the
chemical reaction between the lactic acid and the alkali
(sodium or calcium hydroxide) may have been detrimental
directly to the culture organisms or indirectly by causing
a given amount of heat during drying to be more effective
in destruction of the organisms.

Calcium hydroxide (or its salt) was more detrimental
than the sodium products. Standard plate counts of
neutralized cultures (calcium hydroxide) immediately after
drying were below 300,000 per gram. The cultures neutral-
ized with sodium hydroxide and similar processing condi-
tions had 10 million or more organisms per gram.

The activity of lactic cultures dried from liquid
cultures that were incubated 3, 12, 16 and 2h hours indi-
cated 3 hours of incubation was insufficient for maximum
results. There was not an important difference among the
other three periods (12, 16 and 2h hours) although the

samples from the 16-hour period in all trials showed a

38
slightly higher initial activity.

One explanation for the above results may be the
physiological age. The organisms' ability to survive the
adverse effects of the hot air in drying condition might
be influenced by the age. The organisms from an under-
ripened culture might be more easily destroyed.

Another explanation for the results of the incubation
trials is that the largest number survived (hence the most
activity) on the basis of the largest number in the cul-
tures before drying (other conditions being equal). How-
ever, large differences were observed in total count of
organisms in the dry cultures having initial activities
within a very narrow range. Additional investigations
need to be conducted to elucidate the correct reasons.

In the range of Optimum outlet air drying tempera-
tures, the additions of gelatin in small amounts to the
liquid cultures failed to influence activity of dry
cultures. One must assume the gelatin did not exert a
protective effect against the lethal property of the hot
air during dehydration.

The results of the storage tests, namely diminishing
activity with increasing temperatures and periods, are not
unusual. The same phenomenon occur constantly in nature.
Vegetative cells perish under natural adverse conditions,
particularly prolonged dry periods.

The exact processes that take place in the demise of

39
a bacterial cell under extended periods of dehydration are
not established. Presumably the need for food to maintain
the vital activities of cell life is a function of time
and temperature. Undoubtedly, inherent characteristics of
the species and the individual cell within the same species
affect the rate with which viability is destroyed or the

rate of recovery as indicated by population increase.

 

hO

SUMMARY ahD CONCLUSION

The project was conducted to study the feasibility of
dehydrating cheese cultures by means of the conventional
spray process that is used in the production of dry milks. i a“
The greatest needs in the manufacture of dry cheese cul-

ture are for economy and a rapid rate of growth that is i**

 

maintained for an adequate length of time. The term
"activity" was used to designate growth rate by measuring
acidity production. Activity (expressed as a whole number)
was the percent acidity multiplied by 100, that developed
in sterile skim milk inoculated with 1 percent dry cul-
ture and incubated 16 hours at 720 F.
Results of the study show that generally with favor-
able conditions, cheese culture organisms will survive
syray process dehydration with sufficient viability to
coagulate sterile skim milk with 1 percent inoculation and
incubation for 16 hours at 720 F. The correlation between
outlet air drying temperature and acid producing potential

ivity) of the dry cheese culture vas closer than

('1’

(ac
between inlet air drying temperature and acid producing
potential. Jecreases in outlet air temperature caused
increased initial activity of dry cultures. Cultures
dehydrated with outlet air temperatures below 1650 F. were

more active than those dried above this temperature.

Decreases in acidity of ripened cultures with sodiu:

:‘S

or calcium hydroxide prior to iehydratinr caused corre-

C)

0
q

spending decreases in the activity of the dried cultures.
A comparison of culture incubation times of 3, 12, lb

)1

and ch hours before dehylration showed the least initial
activity in the dried culture from the 3-hour incubation.
only minor differences in cultures were obtained among the
remaining three periods (12, lo and 2h hours); however of
these the most activity (slight) was from the lb-hour
samples.

The addition of 3.05, 0.10, 0.?0 or 1.00 percent
gelatin to skim milk before pasteurization, inoculation,
incubation and dehydration did not seem to affect the
initial activity of the dry culture.

Activities of dchy rated cultures that were stored
1 year at ~lSO F. compared favorably with 'nitial activi-
ties although small decreases occurred. Samples with high
initial activities (70 to 80) that were stored at too F.
had a small average decrease after 1 week and a substantial
decrease after 2 weeks (average was 57). After 3 weeks of
store e, little activity was evident according to the
tests. Dehydrated cultures d monstrated little activity

1 ,‘O ‘N. 0
after storage for l weex at 7: or 70 F.

“ii-m1-

( )

U1

A
(j\
V

(8)

LITERATURE CITED

n

Anderson, T. r. Techniques for the preservation of
three-dimensional structure in preparing speci-
mens for the electron microscooe. E. ;. Acad.
9393;” Ser. II 13: 13o-13ii. 195:1.

 

Appleman, m. D. and Sears, 0. H. Studies on 1y0phil- .
ed cultures: lyophile storage of cultures of P‘
fihizobium leguminosarum. i. g: bact., 52:
209-211. lého.

Drown, J. H. The preservation of bacteria in vacuo.
II. i. of hact., 23: ab. 1932. (Abstract

from thirty-third annual meeting of the Society
of American jacteriologists).

Christian, R. T. The effect of various low pressures
on the percent livability of Serratia marcescens
and Micrococcus pyogenes var. aureus desiccated
from the frozen state. Thesis, Michigan State
University. 1952.

 

Downs, P. A. Bacterial flora of milk powder. Thesis,
Cornell University Library. l920. (Jriginal
not seen - quoted from Supplee).

blser, N. J., Thomas, R. A., and Steffen, G. I. ‘he
desiccation of Sara and other biolo;ical products
(including microorganisms) in the frozen state
with the preservation of the original qualities
of products so treated. l. £mmunol., 23: A33-

Flosdorf, n. J., Hull, L. J., and Judd, S. Drying by
sublimation. i. of Immunol., 50: 21-Sh. lQhS.

damova-fiaukova, N. I. (Research inst. Jairy Milk
lndust., Moscow). Method of preparation of dry
cultures of lactic acid bacteria grown with
growth promoting substances (auxins) in vacuo at
low temperatures. mikrobiologiia, 17: 66-75.
lghd.

A
\Q
v

(11)

M3

Hammer,
bacteria. _
(Original not seen.
Christian).

3. s. A note on the vacuum desiccation of
J. of had. Res., 2): 527-530. 1911.
Quoted by blscr and

H‘35inbottom, C. Bacteriological studies of roller-
dried milk powiers, roller-dried buttermilk and
. , ~ (7 7‘?
of roller and spra ~dried whey. i. uairl
desearch, l3: 3D’-323. lgui.

Higginbottom, C. The effect of storage at different
relative humidities on the survival of micro-
organisms in milk powder and in pure cultures

4 ' m' R J. Jai v Fe ea .h 0:
dgie: 1n 4&1 . _ r, x 3 re ., 2
O/‘7Q' 19)}.

fiunwicke, d. p3 and Jephcott, H. The destruction of
bacteria in the roller process of milk drying.
J. Dairy Sci., 3: ace-alt. 1925.

 

Macy, H. Some observations on the bacterial content
‘ w . -.. o —J ’ A/
of dried mil&. 3. Jairi bc1., ll: bib-520.
1923.

mattick, A. T. 3., Hiscox, L. 3., and Crossley, a. L.
The effect of the various factors upon the
bacterial (plate) count of the intermediate
products and of the final powder. J. Dairy
n a. , 1L. 1on-11) 1.}r “
neseuzch, +- 3) 44. 94).

a. J. The preservation

:1. 2.1.: BaCt°9 33:

Morton, H. h. and Pulaski,
of bacterial cultures.
1' O

 

Naylor, H. B. and Smith, P. A. Factors affecting
the viability of igrratia marcescens during
dehydration and storaée. i. gf Eact. S2:
sea-:73. l9ho.

Kichols, A. A. Bacteriological studies of spray-

 

dried milk powder. Q. ggjrv ”csearch, 1):
Olson, J. 3., Jr., and Nielsen, A. J. Changes in

bacterial counts and flavor of dry milks after
recombination by various means and storage at
low temperatures. i. Dairy Sci., 33: 301-370.

1955.

 

(19}

i u
UK
\..I

Poor,

room conditions
Streptococci, and staphylococci.

3.

AL

(‘1‘

J. and Fithatrick, C. P. .ne destructive
effects of light and drying and other living-

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