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This is to certify that the

thesis entitled

AGGLUTINATION BEHAVIOR OF LACTOCOCCI AS DETERMINED BY
ENZYME LINKED IMMUNOSORBENT ASSAY AND LASER SCANNING
MICROSCOPY

presented by

Christopher Sypien

has been accepted towards fulfillment
of the requirements for

Masters degree in ch 3

   
 

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Date 63,“ ‘ ’48

 

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1M animus-mu

AGGLUTINATION BEHAVIOR OF LACTOCOCCI As DETERMINED BY
ENZYME LINKED IMMUNOSORBENT ASSAY AND LASER SCANNING
MICROSCOPY
By

Christopher Sypien

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1998

ABSTRACT
AGGLUTINATION BEHAVIOR OF LACTOCOCCI AS DETERMINED BY

ENZYME LINKED IMMUNOSORBENT ASSAY AND LASER SCANNING
MICROSCOPY

By

Christopher Sypien

An ELISA was developed as a rapid method to screen lactococci for
agglutination behavior and to determine antibody stability. Bovine antibodies were
isolated by ammonium sulfate precipitation of colostrum secretions. The screening
ELISA results suggested that IgA plays a minor role in agglutination, whereas the
role of lgG might be understated in the current literature. The antibodies were then
either heat treated at 71, 75, 80 or 85°C, for 1 to 90 minutes at 5 minute intervals or
enzyme treated at concentrations of O Ulnl to 135.6 Ulnl of papain. Although
heating to 71°C for 45 minutes was sufficient for pasteurization, effective reduction
of the immunogenicity of the milk antibodies that cause agglutination as determined
by the ELISA was not demonstrated. Increasing the pasteurization temperature to
75 or 80°C for 45 minutes caused decreases (P<0.05) in IgM, lgG and IgA antibody
binding to lactococci. ELISA results indicate that papain represents a method to
cleave antibodies and prevent agglutination. Laser scanning microscopic analysis
using immunofluorescence confirmed the results of the ELISA for the screening,

heat and enzyme procedures.

TABLE OF CONTENTS

List of tables ................................................................................................................... vi
List of figures ............................................................................................................... vii
Introduction ..................................................................................................................... 1
1. Literature review ........................................................................................................ 3
1.1. Lactic acid bacteria as starter cultures ................................................................... 3
1.2. Bovine milk immunoglobulins ................................................................................. 5
1.3. Starter culture agglutination ................................................................................... 7
1.4. Factors affecting agglutination ............................................................................. 10
1.5. Methods to inhibit culture agglutination ............................................................... 12
1.6. Defects associated with bacterial agglutination ................................................... 16
1.7. Agglutinating detection and screening procedures ............................................. 17
2. Materials and methods ............................................................................................ 22
2.1. Cultures ................................................................................................................. 22
2.2. Colostral antibody preparation ............................................................................. 23
2.3. ELISA procedures ................................................................................................. 23
2.3.1. Sample preparation for ELISA .......................................................................... 25

2.3.1.1. Effect of Heat on stability of bovine milk immunoglobulins and
their ability to bind lactococci ....................................................................................... 25

iii

2.3.1.2. Effect of papain and pepsin on stability of bovine milk

immunoglobulins and their ability to bind lactococci ................................................... 25
2.3.1.3. Screening culture for agglutination behavior ................................................. 26
2.4. Laser scanning microscopy .................................................................................. 26
2.5. Verification of agglutination in skim milk .............................................................. 28
2.5.1 Sample preparation for verification of agglutination in skim milk ...................... 29

2.5.1.1. Effect of Heat on the stability of bovine milk immunoglobulins
and their ability to bind lactococci ............................................................................... 29

2.5.2.2. Effect of enzyme treatment on the stability of bovine milk
immunoglobulins and their ability to bind lactococci enzyme study

verification .................................................................................................................... 29
2.6. Statistical analysis ................................................................................................ 30
Results and discussion ................................................................................................ 31
3.1. Screening of cultures for agglutination behavior ................................................. 31
3.1.1. Culture agglutination as determined by the ELISA .......................................... 31
3.1.2. Aggiutination behavior as determined by laser scanning microscopy ............. 36
3.2 Effect of heat treatment on the stability of bovine milk immunoglobulins ............ 42
3.2.1. Antibody stability to heat treatment as determined by the ELISA .................... 42
3.2.2. Antibody stability to heat as determined by laser scanning microscopy ......... 51
3.2.3. Verification in Skim milk ..................................................................................... 56
3.3. Enzyme treatment of immunoglobulins to reduce agglutination ......................... 61
3.3.1. Antibody stability to enzyme treatment as determined by the ELISA .............. 61
3.3.2. Antibody stability to enzyme treatment as determined by

Laser scanning microscopy ......................................................................................... 68
3.3.3. Verification in skim milk .................................................................................... 71

iv

Conclusions .................................................................................................................. 72

Recommendations ....................................................................................................... 74
Bibliography ................................................................................................................. 76
Appendix. Pepsin ELISA and Laser Scanning Microscopy ...................................... 82

LIST OF TABLES

Table 1. Comparison of agglutination behavior of lactococci provided by the
supplier, to the screening method of Hicks and Ibrahim (1992) ................................. 32

Table 2. Ability of single and mixed strain lactococci to bind bovine milk
immunoglobulins as determined by the ELISA ........................................................... 33

Table 3. Heat stability of bovine IgM and its ability to bind lactococci
after heat treatment of 45 minutes ............................................................................... 43

Table 4. Heat stability of bovine IgG and its ability to bind lactococci
after heat treatment of 45 minutes ............................................................................... 46

Table 5. Heat stability of bovine IgA and its ability to bind lactococci
after heat treatment of 45 minutes ............................................................................... 48

Table 6. The pH differentials in 1000 ml cylinders of skim milk fermented
with lactococci grown in media heated at various temperatures ........................ 59

Table 7. The stability of bovine lgM and its ability to bind lactococci
after papain treatment at 37°C for 60 minutes ............................................................ 63

Table 8. The stability of bovine IgG and its ability to bind lactococci
after papain treatment at 37°C for 60 minutes ............................................................ 64

Table 9. The stability of bovine IgA and its ability to bind lactococci
after papain treatment at 37°C for 60 minutes ............................................................ 65

Table 10. The stability of bovine IgM and its ability to bind lactococci after
pepsin treatment at 37°C for 60 minutes ..................................................................... 82

Table 11. The stability of bovine IgG and its ability to bind lactococci after
pepsin treatment at 37°C for 60 minutes ..................................................................... 83

Table 12. The stability of bovine IgA and its ability to bind lactococci after
pepsin treatment at 37°C for 60 minutes ..................................................................... 84

vi

LIST OF FIGURES

Figure 1. Ability of single and mixed strain lactococci cultures to bind
IgM, IgG and IgA as determined by the ELISA ........................................................... 34

Figure 2. Laser scanning micrograph of lactococci treated with bovine

milk immunoglobulins: WWA (a,b), E72 (c,d), SGi (e,f), M30 (g,h).

ace and g = phase contrast; b,d,f, and h = fluorescent images stained

with fluorescein conjugated bovine anti lgG. Magnification 4000x ........................... 37

Figure 3. Heat stability of bovine lgM and its ability to bind lactococci as
determined by an ELISA. Optical density in the unheated sample was
100% ............................................................................................................................ 44

Figure 4. Heat stability of bovine IgG and its ability to bind lactococci as
determined by an ELISA. Optical density in the unheated sample was
100% ............................................................................................................................ 47

Figure 5. Heat stability of bovine IgA and its ability to bind lactococci as
determined by an ELISA. Optical density in the unheated sample was
100% ............................................................................................................................ 49

Figure 6. Laser scanning micrograph of lactococci with heat treated lgG:

unheated control (a,b), heated at 71°C for 45 min (c,d), heated at 75°C

for 45 min (e,f), and heated at 80°C for 45 min (g,h). ace and g = phase

contrast; b,d,f, and h = fluorescence, stained with fluorescein conjugated bovine
anti lgG. Magnification 4000x .................................................................................... 52

Figure 7. Laser scanning micrograph of lactococci with heat treated lgM:

unheated control (a,b), heated at 71°C for 45 min (c,d), heated at 75°C

for 45 min (e,f), and heated at 80°C for 45 min (g,h). ace, and g = phase

contrast; b,d,f, and h = fluorescence, stained with fluorescein conjugated bovine
anti IgM. Magnification 4000x .................................................................................... 54

Figure 8. Laser scanning micrograph of lactococci with heat treated IgA:

unheated control (a,b), heated at 71°C for 45 min (c,d), heated at 75°C

for 45 min (e,f), and heated at 80°C for 45 min (g,h). ace, and g = phase

contrast; b,d,f, and h = fluorescence, stained with fluorescein conjugated bovine
anti lgA. Magnification 4000x ..................................................................................... 55

vii

Figure 9. The effect of heat treatment on culture performance measured by pH
differential. Top and bottom pH decrease in 1000 ml cylinders of skim milk
inoculated with lactococci grown in media heat treated at various

temperatures ................................................................................................................ 57

Figure 10. Effect of papain treatment for 2 h at 37°C on the stability of bovine IgM,
lgG and IgA and their ability to bind lactococci as determined by an ELISA. Optical
density in the nonenzyme treated sample was 100% ................................................ 62

Figure 11. Laser scanning micrograph of commercial culture SG1 grown in

Insure media after papain treatment at: 27.1 U/nl (a,b), 135.6 U/nl (c,d), culture M30
grown in Insure media after papain treatment at 27.1 Ulnl (e,f),

135.6 Ulnl (g,h). ace and g = phase contrast; b,d,f, and h = fluorescent

images stained with fluorescein conjugated bovine anti lgG. Magnification

4000x ............................................................................................................................ 69

Figure 12. Effect of pepsin treatment for 2 h at 37°C on the stability of bovine IgM,
IgG and IgA and their ability to bind lactococci as determined by an ELISA. Optical
density in the nonenzyme treated sample was 100% ................................................ 85

Figure 13. Laser scanning micrograph of commercial culture SG1 grown in

Insure media after pepsin treatment at: 375 Ulnl (a,b), 1,880 Ulnl (c,d), and culture
M30 grown in Insure media after papain treatment at 375 U/nl (e,f),

1,880 Ulnl (g,h). a,c,e, and g = phase contrast; b,d,f, and h = fluorescent

images stained with fluorescein conjugated bovine anti lgG. Magnification

4000x ............................................................................................................................ 86

viii

INTRODUCTION

The occurrence of bacteriophage, antibiotic residues, sanitizers, cleaning
agents and agglutination are all problems associated with the manufacture of
Cottage Cheese (Ryan, 1985). Aggiutination is increasingly being recognized for its
effects on starter performance, however some Cottage cheese manufacture’s still do
not fully appreciate the magnitude of the agglutination problem (Hicks and Hamzah,
1992). Therefore agglutination represents an unrecognized revenue loss to the
dairy industry.

Aggiutination is the result of an antibody-antigen reaction (Salih and Sandine,
1984), in which antibodies naturally present in milk bind to the surface cell receptors
of bacteria. The antibodies being multivalent, join the bacteria together into large
chains and clumps. The clumps grow in size until a critical mass is reached at which
time the bacteria and casein precipitates to the bottom of the vat, producing yield
and quality problems during Cheese manufacture. Aggiutination problems were first
recognized in cheeses which use long incubation periods. However, Salih and
Sandine (1984) observed the agglutination problem as soon as one hour after
inoculation; and it has been recently suggested that agglutination may be a problem
in other cultured dairy products that employ lactococci (Hicks and Milton, 1990;

Ustunol and Hicks, 1994).

Many of the methods suggested to prevent the agglutination of starter
bacteria have not been entirely successful or practical. Thus, there is a need for
further inquiry into developing preventive measures for this problem. In light of
recent advances in immunological techniques, the problem of immunoglobulin-
bacterial interactions during fermentation’s can be directly studied, with the ultimate
goal of developing better preventative measures. The objectives of this study were:
1) To develop a modified Enzyme Linked Immunosorbent Assay (ELISA) to screen
cultures for their agglutination characteristics; 2) Investigate the stability of bovine
immunoglobulin to heat or enzymes using an ELISA; 3) Verify the ELISA using
Laser Scanning Microscopy (LSM); and 4)Verify the stability ELISA results in a skim

milk system.

1. Literature review

1.1. Lactic acid bacteria as starter cultures

Lactic acid bacteria have multiple functions when used as starter organisms
for dairy products. The lactics are gram positive rods or cocci, which are nonmotile,
nonpigmented, catalase negative, and facultative anaerobes (Axelsson, 1993).
These bacteria ferment sugars, mainly lactose, to produce lactic acid and other
compounds; and play a role in protein and lipid hydrolysis which contributes to the
taste and texture Characteristics of dairy products.

Starter cultures for dairy products may consist of one strain (single
organisms) or multiple strains (blends of defined strains) or traditional mixed strains
(undefined numbers and ratios of strains) of several organisms. These cultures are
used with or without rotation depending on the manufacturer characteristics (Mayra-
Makinen and Bigret, 1993). The pathway used to ferment lactose and produce lactic
acid represents a distinguishing feature for classification of lactic acid bacteria.
Using the Embden-Meyerhoff pathway, the homoferrnenters convert glucose —> 2
pyruvate —> 2 lactate. Whereas, the heterofermenters convert glucose —9 gluconic
acid ——> C02 and pentose which is then converted to ethanol + lactic acid using the
Hexose-Monophosphate pathway (Salminen and Wright, 1993). The main

difference is that the homoferrnenters produce lactic acid in a 2:1 ratio when

compared to the heterofermenters. Use of homofennenter starter cultures are
desirable in the production of many fermented foods, especially dairy products such
as cheese where lactic acid is the desired product of the fermentation. The dairy
industry relies heavily on the use of lactic acid bacteria as starter cultures. Some
products include but are not limited to cheese, yogurt, sour cream, cultured milk, and
cultured butter. In fact lactic acid bacteria can be used to produce over 1000
different products each with its own texture and flavor characteristics (Mayra-
Makinen and Bigret, 1993).

A major concern for dairy manufacturers has been bacteriophage, which has
been the main cause of starter failure (Lawrence, 1978). These bacterial viruses
are strain and species specific, in that they can only attach and inject genetic
material into those bacteria which have the specific attachment Site. The phage
insert their genetic material into the DNA of the bacteria. After replication and the
production of viral proteins the cells lyse and multiple phage are released to infect
other cells (Kuby, 1992). For this reason a great deal of research has been
conducted in methods to inhibit phage infection and the identification of phage-
resistant cultures. Other agents which cause batch failure include improperly used
cleaning agents, sanitizers, as well as, antibiotics which kill the starter culture before
it completes the fermentation. Another factor in starter culture performance is the

interaction between immunoglobulins naturally present in the milk or media and the

starter culture. This interaction, known as agglutination, is increasingly being

recognized for its effect on dairy starter cultures.

1.2. Bovine milk immunoglobulins.

The globular proteins known as immunoglobulins are the first line of defense
against bacteria and viruses (Kuby, 1992). Some strains of lactococci have been
known to auto agglutinate, however antibody titers (Ellenhan, 1975; Emmons et al.,
1966) and the frequency that the antigenic determinant is expressed on the cell
surface are thought to be the major factors causing agglutination (Scheuble et al.,
1989). lgA, lgG, and lgM are the three main classes of immunoglobulins identified
in bovine milk, and colostrum (Butler, 1983).

lgG is the most abundant immunoglobulin found in bovine milk. lgG is found
as a monomer in milk secretions and has a molecular weight of 150,000. lgG
consists of two heavy chain and two light chain components (Kuby, 1992).
lnterchain disulfide bonds hold the heavy and light chains together forming a "Y"
shape. The monomer consists of two fragments, the tail or Fc region and the arms
or Fab fragments. The lgG immunoglobulin class consists of two subclasses of
antibodies lgG1 and Ing. lgG is the major antibody found in colostrum
representing as much as 80% of the whey protein component of colostrum, whereas
Ing is found at a level 40 times lower (Mach and Pahud, 1971 ). Although the

amount of total antibodies is reduced in normal milk when compared to colostrum,

IgG1 is still the predominant antibody secreted by the mammary gland (Butler, 1983).
The immunoglobulin lgA is found as dimer with a disulfide bond holding two
monomers together. This type of configuration allows for four possible antigen
binding sites instead of the normal two found with a single monomer. The dimeric
form of IgA gives it better access to antigens increasing its agglutination ability.
Bovine secretions contain very large amounts of secretory lgA (SlgA). The
secretory piece transports the immunoglobulin across the mucous membrane and is
thought to provide protection from proteolytic enzymes. In the past, researchers
have not included lgA as a contributing factor in the agglutination behavior of
lactococci because of its relatively low quantities in bovine milk. In spite of the low
levels, lgA will bind lactic cultures and could be a hidden factor during agglutination.
lgM is found in a pentamer configuration consisting of five, four chain polymers
which are linked together through disulfide bonds on the FC portion of the
immunoglobulin. This type of configuration allows for ten possible binding sites and
iS the reason that IgM remains the most effective agglutinating antibody (Rose et al,
1964).

In cows milk, immunoglobulins represent 0.08% of total milk protein and 3%
of milk whey (Mach and Pahud, 1971). Colostrum, which is the first milk secreted
after calving, contains eighty times more lgG and lgM than normal milk (Salih and
Sandine, 1984). Milk contains 0.7 mglml of bovine lgG, however in colostrum lgG

levels can reach 100 mglml, with levels generally returning to normal within a week

of calving (Butler, 1983). The large supply of antibodies in colostrum provides
passive immunity to the newborn because the fetal calf does not acquire immunity in
utero (Brambel, 1970). Emmons et al. (1963) reported that immunoglobulins are
always present in bulk milk and it is suggested that the agglutination problem may
also be partially due to contamination of bulk milk with colostrum. In Canada, as in
many countries, colostrum is not permitted in the milk supply. However, Zawistowski
and Mackinnon (1993) found that 89% of the samples tested in Canada were
contaminated with colostrum.

In 1976, Kanno et al. isolated crude "euglobulin" fraction of milk by gel
filtration and tested the agglutination activity of the different fractions against
Lactococcus lactis ssp. cremon's strain HP. Of the three fractions, lgM was shown
as the main factor causing agglutination. They also determined that the activity of
agglutination was maximized in the pH range of 6.5 to 8.5. Salih and Sandine
(1984) showed, using fluorescent techniques, that lgG bound more frequently than
IgM. Although lgG is found in greater quantities, lgM with its pentameric structure is

thought to be the primary agglutinating factor (Kanno et al., 1976).

1.3. Starter culture agglutination
Acid coagulated cheeses are manufactured by precipitation of the milk
proteins as the pH is lowered (by acidification) until the isoelectric point is reached.

The average isoelectric point for casein micelles is approximately 4.6 at which time

the casein micelle unfolds and a protein network is formed trapping fat and moisture
in the matrix. Acidification can occur in two ways, the first is direct addition in which
food grade acid is added directly to the vat. The second method involves the use of
starter cultures in which bacteria produce the acid that ultimately leads to
coagulation. Any factor that inhibits culture performance can have a consequence
on acid production, thus coagulation, effecting both the yield and the quality of the
product.

The ability of certain substances naturally present in milk to interfere with
coagulation was first shown by Stocking (1904). Aggiutination is the result of an
antigen-antibody reaction as shown by Salih and Sandine (1984). This problem is
the result of agglutinins (immunoglobulins) naturally present in milk binding to the
surface cell receptors or epitope’s of the starter bacteria. The antibodies being
multivalent can bind to multiple bacteria causing them to aggregate and forming
chains or clumps. The bacteria in these clumps continue to produce acid which
precipitates the casein’s around the bacteria and adds to the weight of the complex.

When a critical mass is reached, the cells begin to sediment, (Milton et al., 1990)
causing uneven distribution of the bacteria in the vat and thus uneven acid leading
to yield and quality problems in production (Milton et al., 1990). This was also
observed by Weeks et al. (1994) who reported the same settling of bacteria during

heavy coagulum formation.

A closely related phenomena to agglutination is aggregation which occurs for
conjugation purposes. The aggregation of cells greatly enhances the ability for
effective transfer of genetic information. A study by Wang et al. (1994) Showed that
the efficiency of genetic transfer was 102 — 105 times higher for bacterial cells that are
able to self aggregate. Upon PAGE analysis it was determined that aggregation
positive cells contained a unique protein 125 kDa in size. Under these
circumstances cell aggregation is a positive adaptation for lactic acid bacteria.
Unlike cell aggregation, agglutination, however, is a negative aspect which results in
a decrease in acid production and loss revenues due to decrease quality and yield.

Morice et al. (1991) found that Streptococcus thermophilus will grow in
shorter chains in the presence of cations such as an“, Ca2+ Mg”, Fe”, and Zn”.
They determined that sodium had a shorting effect in 17 out of 20 strains of
Streptococcus thermophilus with best reductions occurring with sodium
concentrations over 80 mM. They also concluded that the anion does not play a
role in the dechaining activity of sodium salts.

Weeks et al. (1994) reported the occurrence of a layer of heavy coagulum
containing high total solids on the base of commercial lactic casein silos. They
termed this phenomenon heavy coagulum formation and concluded that this
occurrence was similar to that proposed by Emmons et al. (1966) for minor sludge
formation in Cottage cheese. Commercial lactic casein manufacture differs from

Cottage cheese manufacture by the size of the coagulation vessel and the length of

incubation. The incubation for Cottage cheese is roughly 5-6 hours, which is
considerably less than that of the 14-16 hours needed for lactic casein manufacture.
Yield loss is not the major concern in heavy coagulum formation due to an efficient
fines recovery system. However, the variation produced creates difficulty during
processing and requires a blending step. Therefore, the elimination of heavy

coagulum formation as agglutination would greatly benefit the dairy industry.

1.4. Factors affecting agglutination

Emmons et al. (1963) observed that immunoglobulins are always present in
bulk milk. Bovine milk contaminated with colostrum favors agglutination; but milk
that has been homogenized or pasteurized at higher than normal temperatures,
reduce agglutination (Emmons et al., 1966). The degree of agglutination is
dependent upon the strain of bacteria and the agglutination titer (Scheuble et al.,
1989). The variability between strains is the result of the number of epitopes found
on the surface of the bacteria. The strains of bacteria that contain more epitopes will
be more prone to binding antibodies and thus more to agglutination. However,
Emmons et al. (1966) suggested that it was the antibody titer which is most
important in determining the severity of agglutination.

Emmons et al. (1966) also reported that chain length increased the extent of
settling. Scheuble et al. (1989) observed no significant difference based on chain

length in cultures that agglutinate severely, compared to those that do not

10

agglutinate at all. Shorter chains of bacteria decreases the consequences of
agglutination as they will lack the critical mass necessary to sediment and will be
more evenly distributed in the vat. The cultures that grow in longer chains upon
agglutination will reach the critical mass quicker and thus the extent of settling will
occur sooner and to a greater extent. Ibrahim (1995), reported that the proteolytic
activity of starter cultures might be a clue into the agglutination behavior of bacteria.
He reported that the proteolytic activity of agglutination sensitive cultures was lower
than that of agglutination resistant cultures. The amount of agitation that occurs
may have a pronounced effect upon agglutination severity. Prolonged agitation was
once thought to increased acid production and decreased culture agglutination in
skim milk (Gillies, 1959). However, Emmons et al. (1966) documented a decrease
in acid production and an increase in culture agglutination upon prolonged agitation.
Gosling et al. (1995a) recently reported that agitation during coagulation could
either increase or decrease heavy coagulum formation depending on the timing and
geometry of agitation.

Antibodies are naturally present in whey based media, which are typically
used to grow lactic starter cultures for vat inoculation. For that reason, the bacteria
could agglutinate in the bulk starter media before being added to the vat. In severe
cases, batch failure can occur if the lactococci has already agglutinated in the media

before addition to the vat.

11

1.5. Methods to inhibit culture agglutination

Chemical denaturants such as 2-mercaptoethanol can denature antibodies
by dissociating the disulfide bonds allowing for separation of the heavy and light
chains. Kanno et al. (1976) saw 76%-86% reduction in agglutination activity upon
treatment with 0.2 M 2-mercaptoethanol for three hours at room temperature or one
hour at 30°C. Surfactants have been used to decrease chain formation (Hicks and
O'Leary, 1986). It was once believed that lecithin may hinder attachment of
antibodies by changing or coating epitopes on the cell surface (Stollerrnan and
Ekstedt, 1957). Recently, Milton et al. (1990) determined by cell counts that
increases in colony forming units occurred when low concentrations of lecithin were
used but then a decrease in agglutination as concentrations were increased. They
observed the greatest increase, 0.3 log“), when 0.5% lecithin treatment was used
and than a decrease in bacteria counts as the treatment levels were increased to
1% and 2%. The use of lecithin could increase agglutination by facilitating chain
fragmentation (Milton et al., 1990).

Enzyme Cleavage also represents a way to destroy antibodies. The enzyme
papain is a sulfhydryl protease isolated from Canoe papaya, and has a molecular
weight of 23,000. After brief exposure to the antibody, the enzyme papain will
cleave the most susceptible bonds. Papain has nonspecific activity and will digest
the entire protein if given proper time. The most susceptible bonds on an lgG

immunoglobulin are on the heavy chain below the lnterchain disulfide bonds. After

12

digestion, this leaves two Fab fragments which may still bind to the antigen and a Fc
fragment (Kuby, 1992). Ustunol and Hicks (1994) suggested that Fab fragments
from papain digestion will bind to the antigenic binding sites on the bacteria and
prevent further agglutination interactions by blocking fully intact immunoglobulins
present either in the media or milk. In their study, antibodies from whey fractions
were digested with papain during ultrafiltration and diafiltration to isolate those
antibody fragments with a molecular weight less than 10,000 Da. These fragments
were then freeze dried and added to an internal pH control buffer salt mixture.
Commercial cultures OS, M30, and M37 when grown in this reconstituted media
Show pH differential reductions of 40, 55 and 72% for the cultures OS, M30 and M37
respectively. The enzyme pepsin will also cleave antibodies, however unlike
papain, pepsin cleaves the bonds just below the interchain disulfide bonds. On an
lgG molecule pepsin produces one main fragment called F(ab)2 fragment and many
small fragments that composed the Fc fragment. The F (ab); fragment consists of
the two Fab fragments joined by interchain disulfide bonds. The joining still allows it
the capability of precipitating the antigens (Kuby, 1992).

The use of heat to inactivate antibodies was first shown by Emmons et al.
(1966). They reported eliminating the agglutination activity in milk by heating at
71°C for 30 minutes. Dominguez et al. (1997) determined that no loss in antigen
binding activity of lgG occurred after 60 minutes at 65°C. Lindstrom et al. (1994)

concluded that thermal treatment of immunoglobulin will cause the antibodies to

13

unfold and aggregate. They reported that the unfolding of bovine antibodies due to
heat is irreversible and not very dependent on pH as determined by differential
scanning calorimetry. De Wit and Klarenbeek (1984) reported that lgG unfolds at
72°C in 0.7 M phosphate buffer, with values extrapolated to a heating rate of
21 .4° K/min. Fukumoto et al. (1994) who using radial immunodiffusion reported D
values of 329 minutes at 70°C, 38.8 minutes at 74°C, and 4.3 minutes at 80°C.
Dominguez et al., (1997) reported that at 72°C it took only 1387 seconds for a 90%
reduction in the binding activity of IgG and suggested that the ability of IgG to bind
antigens after heat treatment probably has been overestimated in the literature. Li-
Chan et al. (1995) reported Slightly higher D values for lgG than Dominguez et al.,
(1997). Li-Chan et al. (1995) concluded that a substantial portion of the antibody
activity is retained in milk after commercial processing. Lindstrom et al. ( 1994)
reported that immunoglobulins are among the most heat stable of the whey proteins
and concluded that a temperature of 80.9 1 004°C was needed for the thermal
unfolding of the immunoglobulin fraction of whey. This was consistent with Ruegg et
al., (1977) who determined that the thermal unfolding of immunoglobulins was at
81°C in Simulated milk ultrafiltrate. Later, De Wit et. al. (1983) reported that the
immunoglobulin fraction denatured at 79°C.

Homogenization of bulk starter culture has been investigated as a method to
inhibit culture agglutination by Milton et al. (1990) and Russell-Campbell and Hicks

(1992). A homogenization pressure of 246 Kg/cmz was sufficient to Inhibit

l4

agglutination in milk by mechanically breaking apart the Chains of starter cultures
and reducing the critical mass. The homogenization of milk also has been
investigated, and it is believed that the homogenization of skim milk causes the skim
milk membrane to conform and expose antibody binding sites allowing antibodies to
bind the skim milk membrane instead of the lactic starter (Russell-Campbell and
Hicks, 1992). Russell-Campbell and Hicks (1992) determined that as the
homogenization pressure is increased, more antibody binding Sites become
available on the skim milk membrane, leading to a decrease in agglutination
tendencies of the culture. For this reason, they recommended that skim milk used
for Cottage cheese manufacture be homogenized at a pressure of at least 176
kg/cm2 to reduce agglutination. However, the use of homogenization to prevent
agglutination increases equipment expenditures, the labor requirements, and
ultimately the cost of production.

Similar to homogenization, the use of colloid milling was investigated by
Gosling et al. (1995a) in which a colloid mill was used to reduce heavy coagulum
formation. They reported that homogenization is an unlikely solution for heavy
coagulum formation due to high capital costs and low throughput of homogenizers.
Colloid mills are Cheaper and have a greater throughput. When used at the proper
time, colloid mills caused complete elimination of heavy coagulum. The main
drawback to this technology is that the milling step needs to take place at 11-12

hours to reduce heavy coagulum formation at which point the incubation continues.

15

The timing of this milling step, however, may create logistical problems for

manufacturers.

1.6. Defects associated with bacterial agglutination

Aggiutination causes two major defects in cultured dairy products. The first
occurs if the clumps of bacteria are suspended in the cheese milk as coagulation
occurs, thus trapping the bacterial casein complex before it sediments to the bottom
of the vat. This results in a grainy defect in the curd upon cutting (Emmons et al.,
1963; Emmons et al., 1966). The second defect is more severe and occurs when
the bacterial complexes aggregate at the bottom of the vat to produce a "sludge
layer." In its severest form, agglutination will prevent milk from coagulating, leaving
the vat unusable. The sediment or Sludge layer may have a slight brown color which
indicates that casein from the Skim milk was precipitated by the complex of cells.
Even minor sludge formation associated with agglutination may be routinely
responsible for yield losses of 4-8% (Gradison et al., 1986). Slow uneven acid
production, grainy curd, sediment formation, along with an easily shattered curd
after cutting, produce inconsistent and poor quality cheese (Emmons et al., 1966).
These defects result in decreased profits for dairy manufacturers by limiting yield

and quality.

16

1.7. Aggiutinating detection and screening procedures

Under optimum conditions, lactic starter culture and thus acid production
Should be uniform throughout a cheese vat during fermentation. Bacterial
agglutination is characterized by uneven acid production where the pH at the bottom
of the vat is typically lower than the pH at the top of the vat due to the settling of
starter bacteria. For this reason, the pH differential between the top and bottom pH
has been used to determine bacterial agglutination by a number of researchers
(Emmons et. al., 1963; Emmons et al., 1966; Salih and Sandine, 1984; Milton et al.,
1990 and Ustunol and Hicks, 1994). Typically, the greater the pH differential, the
greater the severity of agglutination. Milton et al. (1990) used a pH differential
greater than 0.2 as an indicator of culture agglutination, whereas Hicks and Ibrahim
(1992) used 0.09 units and Ibrahim (1995) reported 0.1 units to indicate culture
agglutination. Other researchers have reported that pH measurements were an
unreliable method to determine or monitor heavy coagulum formation (Gosling et al.,
1995b)

Due to the settling of bacteria, microbial enumeration can also be used as an
indicator of agglutination (Emmons et al., 1966; Milton et al., 1990). If culture
agglutination has occurred, the numbers of bacterial cells will be greater on the
bottom of the vat. For that reason, standard plate count or direct microscopic
examination has been used as a simple, rapid test for bacterial agglutination

(Ibrahim, 1995). This is based on the idea that agglutination sensitive cultures

17

appear in long chains and clumps, whereas agglutination resistant cultures are
found in smaller chains with little or no clumping.

Another characteristic of severe agglutination would be the sludge or
sediment found on the bottom of the vat. By measuring the depth of the sediment,
one can obtain a determination of agglutination severity. This can be performed by
direct visual measurement or using a pH meter. Since the sediment contains a
lower pH due to the high concentration of lactic bacteria, the sediment layer can be
measured by the pH drop that occurs when the electrode is inserted into the
sediment (Hicks and Hamzah, 1992). Another method to detect agglutination is the
comparison of top and bottom total solids. As bacteria sediment and deposit on the
bottom of the cheese vat, total solids will increase. When agglutination is occurring,
the total solids from the bottom of the vat will be greater than the total solids from the
top. For commercial Cottage cheese vats, Hicks et al. (1989) determined that total
solids was a more effective and sensitive method to determine culture agglutination
than either pH differentials or bacterial enumeration. They concluded that a
difference of 0.07% between top and bottom total solids can be used as an indicator
of slight agglutination. This was later supported by Gosling et al. (1995b) who
reported that measuring total solids in a suitable coagulation tube is an effective
method to screen cultures for heavy coagulum formation. They reported that this

method is more quantitative and, thus, more objective than other screening tests.

18

Aggiutination can also be detected using fluorescent techniques. Salih and
Sandine (1984) used anti-IgM, and anti-lgG with a fluorescein tag to assess
agglutination. Scheuble et al., (1989) also demonstrated fluorescent techniques by
staining lactic cells with laser grade rhodamine 123, which is a fluorescent cationic
probe taken up selectively by bacterial cells. They incubated the cells with colostral
whey proteins and viewed them under an epifluorescent microscope and confirmed
that actual cells were involved in the agglutination reaction.

The methods used to determine which bacterial strains will agglutinate have
not been entirely successful. Most commercial starters consist of mixed strains,
which may contain both agglutination resistant and agglutination susceptible
bacteria. Auclair and Vassel (1963) found that many cultures, although classified as
resistant, may contain agglutination sensitive strains which become dominant upon
extended subculturing in autoclaved milk. The scenario also exists for bacterial
agglutination to play a larger role in the quality and yield of a product if the resistant
strain is eliminated by phage.

Several researchers (Emmons et al., 1965; Salih and Sandine, 1984; and
Ellenhan, 1975) developed procedures to test strains for agglutination resistance or
sensitivity. Emmons and Ellerman's tests were not completely effective and they
were time consuming. Emmons et al. (1965) developed a test that used a
stereomicroscope to visualize agglutination; however, the endpoint determination for

their test is rather subjective. Ellerman's test involved using microtiter plates, in

19

which bacteria and whey samples were incubated for 18-20 hours in round bottom
wells. Those cultures that did not agglutinate would settle to the bottom of the wells
and form a pellet, and conversely cells that did agglutinate would spread out and
form a thin layer, thus the pellet would be larger than the nonagglutinating culture.
This test relies on the operator to determine the endpoints, making it a subjective
test as well.

A rose bengal stain assay has been developed for the rapid screening of
Bruce/Ia agglutinins. Salih and Sandine (1984) used this information to develop a
procedure to screen for culture agglutination. The procedure requires the
researcher to harvest the cells using a centrifuge and then heat inactivate in a 60°C
water bath for one hour. After centrifugation and dilution, the cells are stained with
rose bengal stain. The procedure requires the researcher to place a drop of stained
cells and a drop of whey on a Brewer‘s diagnostic card. The cells and whey are
then mixed together with a sterile toothpick and the plate is shaken for 4 minutes to
allow for maximum agglutination. The agglutination of cells is determined by looking
at the reaction of stained cells and whey. A positive result has large, clear
aggregate of clumping cells and negative result has no clumping. This procedure
was then modified by Scheuble et al. (1989) to decrease antigen preparation time.
The problems associated with this assay are test time, accuracy, reliability,
subjectivity in determining the endpoints, and application to a plant setting. In spite

of these deficiencies, Thunell et al. (1984) used such test routinely in Cottage

20

cheese plants to identify and replace agglutination sensitive cultures with
agglutination resistant cultures and effectively reduced agglutination.

Screening procedures developed to date may not be entirely effective or
practical. This is due to the long time requirements involved for the assay, and the
subjectivity in interpreting the results. For these reasons, the development of a
rapid, nonsubjective test is still needed. The use of ELISA's in the food industry has
received much attention in recent history. The ELISA is an objective, sensitive,
rapid, simple test that does not require extensive training as many other tests do.

The elimination of agglutination could save millions of dollars in the Cottage
cheese industry. Gradison et al. (1986) suggested that minor sludge formation
might be routinely responsible for yield losses of 4-8% This represents a
considerable amount when results for 1996 showed that over 360 million lbs of
creamed Cottage cheese and 329 million lbs of low fat Cottage Cheese were
produced (National Agricultural Statistics Service, 1996). A conservative estimate of
a 1% increase in Cottage cheese yield would approximately represent an additional
3,600,000 lbs of creamed, and 3,290,000 lbs of low fat Cottage cheese annually.
The increase in yield and quality would represent about $7.5 million to the Cottage
industry alone. Researchers have suggested that agglutination may also be a
problem in other dairy products that use lactococci as starter cultures (Milton, 1990;

Ustunol and Hicks, 1994; Weeks et al., 1994).

21

2. Materials and Methods

2.1. Cultures

Frozen multiple strain commercial cultures of Lactococcus lactis subs. lactis
and Lactococcus lactis subsp. cremon's (M30, M27 and SG1) were provided by
Rhone-Poulenc (Marschall Div., Madison, WI). Single strain cultures WWA, M52,
B62, E72, 478, and 874 were obtained from Chris Hansen's Laboratory (Milwaukee,
WI) and were chosen because of their different agglutination characteristics. All
cultures were stored at -80°C until use.

Both single and multiple strain cultures were screened using the method of
Hicks and Ibrahim (1992). Briefly this procedure involves inoculating the cultures at
a 5% (v/v) level into reconstituted nonfat dry milk (NDM)(10% (wlv) solids
autoclaved 10 min at 121°C and stored at 4°C until use). The inoculated NDM was
incubated for 18 h at room temperature(~ 23°C). Each culture was then inoculated
into 50 g reconstituted NDM as prepared above and incubated for 18 h at room
temperature (~23°C). The 50 g of culture were then added at a 5% (w/v) level to
950 g of pasteurized (63°C for 30 min) skim milk in sterile (15 min at 121°C)1000 ml
graduated cylinders. The cylinders were incubated in a 30°C water bath for 5 h.
Aggiutination was determined visually at the end of the 5 h incubation. If sediment

was observed at the bottom of the cylinder, it was scored as positive (+), whereas

22

cultures with no visible sediment were scored negative (-). This procedure was
replicated 3 times with cultures having three negative replications (-l-/-) Classified as
resistant, three positive (+/+/+) classified as sensitive, and 1 (+/-/-) or 2 (+/+/-)

positives classified as having moderate agglutination behavior.

2.2. Colostral antibody preparation

Bovine colostrum was obtained from the Michigan State University Dairy
Farm. Antibodies were purified by ammonium sulfate precipitation Of colostrum. A
33% solution of ammonium sulfate was added to colostrum, and centrifuged (3,000 x
g) for 20 min. The isolate was redissolved in an equal volume of 0.01M phosphate
buffered saline (PBS; 32.0 g NaCI, 4.99 g N32HPO4, 1.029 g N32PO4, in 4 Liters of
DHZO, pH 7.4) The centrifugation process was then repeated 2x The concentrate
was then dialyzed for 3 days against PBS at 4°C with daily changes of PBS. The

antibodies were aliquoted and frozen at -80°C.

2.3. ELISA procedures

The cultures were propagated overnight at room temperature (~23°C) in
Ellikers broth (Difco, Detroit, MI). The cultures were then heat killed in boiling water
for 2.5 h. The cells were centrifuged (3,000 x g) for 1 min. The pellet was
resuspended in 1ml of sterile PBS. The washing was repeated three times in PBS,

with the final pellet being diluted to give an optical density reading of 14%

23

transmittance at 590 nm (Spectronic 1001, Spectronics Instruments Inc, Rochester,
NY) in 0.1M carbonate-bicarbonate coating buffer (1.59 g Na2C03, 2.93 g NaHCOa,
0.2 g NaN3, in 1 liter of DHZO, pH 9.6). Immunolon 4 plates (Dynatech Laboratories
Inc., Chantilly, VA) were coated with 50 pl per well of either culture or control, and
wrapped in parafilm, aluminum foil, and incubated overnight at 4°C. The plates were
then washed 3 times in 0.02% PBS Tween-20 (PBS-T) (Sigma Chemical Co., St.
Louis, MO), and then blocked with 300 pl of 1.0% Gelatin type A from Porcine skin
(Sigma) for 30 min and washed 4 times in 0.02% PBS-T. The plates were incubated
at 37°C for 1 h with 50 pl of antibody sample per well, prepared as described below.
After washing 5 times in 0.02% PBS-T solution, the plates were incubated for 1 h at
37°C with 100 pl of either Anti-lgG (0.25 ng), Anti-lgA (1.0 ng), or Anti-lgM (2.0 ng)
(Bethyl Laboratories, Inc., Montgomery, TX) diluted in the blocking solution. After
washing 6 times, peroxidase binding was determined using 2,2’Azinobis(3-
ethylbenzo-thiazoline—sulfonic Acid) (ABTS)(Sigma) (ABTS)-H202 substrate as
described by Pestka et al. (1982). Absorbence at 405 nm was read using a ELISA
plate reader (Molecular Probes, Inc., Eugene, OR.) using Softmax software. A
larger optical density was the result of more antibodies binding to lactococci
whereas a lower optical density represented fewer antibodies bound. The ELISA
results for the stability of bovine antibodies IgM, lgG, and IgA were converted to

percents by the following formula: % control 00 = 100 -[ 100(OD control - 00

24

treatment) / OD control]. The zero time on the heat treatment graphs was when the

media was brought to temperature, with all control bindings being 100%.

2.3.1. Sample preparation for ELISA

2.3.1.1. Effect of heat on stability of bovine milk immunoglobulins and their
ability to bind lactococci

Insure media (Galloway West C0,, Fond Du Lac, WI) buffer salts was
reconstituted according to the manufacturer’s directions. Colostral antibodies were
diluted 1:5 (v/v) into Insure media for a final volume of 50 ml and heat treated in
stirred water baths. Samples were taken at 0, 1, 5 min and every 5 min interval
thereafter up to 90 min at 71°C and 75°C. The time intervals were the same for 80
and 85°C, except the last samples at this temperature were taken at 60 min and 30
min, respectively. Samples were immediately placed on ice and stored at 4°C until
use in the ELISA the next day. The ELISA to determine heat stability of IgM, lgG,
and lgA and their ability to bind lactococci was conducted as described previously in

Section 2.3.

2.3.1.2. Effect of papain and pepsin on stability of bovine milk
immunoglobulins and their ability to bind lactococci

Insure media (Galloway West Co.) buffer salts were reconstituted according
to the manufacturer’s directions. Colostral antibodies were diluted 1:5 (v/v) into

Insure media for a final volume of 50 ml and treated either with papain (Sigma) or

25

pepsin (Sigma). Five concentrations of enzyme solution 0%, 5%, 10%, 15%, 20%,
and 25% (v/v) were chosen from preliminary studies. These concentrations when
converted to enzyme activity units (U) which correspond to 0 Ulnl, 27.1 Ulnl, 54.3
Ulnl, 81.3 Ulnl, 108 Ulnl, 135.6 U/nl for papain and to 0 U/nl, 375 Ulnl, 750 Ulnl,
1,100 Ulnl, 1,500 U/nl and 1,875 Ulnl for pepsin. The concentrations were acquired
by adding a saturated solution of papain (0.659 in deionized water) or pepsin (1.50 g
in 3 ml deionized water) to the 1:5 antibody/media mixture. The antibody/media
enzyme solution was incubated at 37°C for 2 h (1 h in a oven, and 1 h as the sample
in the ELISA described in Section 2.3). After incubation, the enzyme and any
unattached antibodies were washed out of the ELISA plate during the washing
procedure. The ELISA to determine enzyme stability of IgM, IgG, and lgA and their

ability to bind lactococci was conducted as described previously in Section 2.3.

2.3.1.3. Screening culture for agglutination behavior
Colostral antibodies were diluted 1:5 (v/v) into 1.0% gelatin blocking solution
for a final volume of 50 ml. The ELISA to determine the amount of IgM, lgG, and lgA

able to bind lactococci was conducted as described previously in Section 2.3.

2.4. Laser scanning microscopy
The single and multiple strain lactococci were grown in Insure media
(Galloway West Co.) overnight until a pH of 4.6 to 5.2 was reached. 240 pl of

culture were centrifuged (3,000 x g) for 1 min. The pellet was resuspended in 160 pl

26

and then washed and resuspended in 80 pl, washed and resuspended in 2 pl of
PBS and allowed to air dry on a slide. The culture smear was circled with a
Peroxidase-Antiperoxidase-Peroxidase (PAP) pen (Research Products lntemational
Corp, Mt. Prospect, IL) to create a well. Fixing agent (ethanol, chloroform, formalin
6:321 ratio) was added to the slides. After 7 min the slides were rinsed with 95%
ethanol. After rinsing with distilled H20, the slides were heat fixed by passing
through an open flame. Previously prepared colostrum antibodies (50 pl) as
described in Section 2.3.1., were added and allowed to react for 15 min at room
temperature (~23°C). Excess antibodies were then washed off the slide with PBS.
Fluorescein conjugated antibodies were diluted in PBS with either a 1:50 (vlv) ratio
for anti-lgG fluorescein (2.0 ng/pl), 1:15 (v/v) anti-IgM fluorescein (5.5 ng/ul) or 1:15
(vlv) anti-lgA fluorescein (6.7 ng/pl) antibodies (Bethyl Laboratories, Inc.,
Montgomery, TX.), and incubated another 15 min on the slide. The slides were
washed with PBS, deionized H20 and air dried. After applying mounting fluid (Difco)
and a cover slip, the slide was viewed using a Zeiss 10 Laser Scanning Confocal
Microscope (Carl Zeiss Inc., Jena, Germany). The 40x oi I objective with a zoom of
100x was used with the contrast set at 820 units for the transmitted images and 353
units for the fluorescent images. Brightness was set at 180 units and 457 units,
respectively on the computer monitor for the transmittance and the fluorescent
pictures. The LSM pictures presented are split screen images in which the right side

is a phase contrast image and the left side is a fluorescent image of the same frame.

27

2.5. Verification of agglutination in skim milk

Skim milk was obtained from the Michigan State University Dairy Plant.
Aggiutination in skim milk was determined by inoculating fermented media at a 5%
level (vlv) into pasteurized (63°C, 30 min.) skim milk contained in a sterile 1,000 mi
graduated cylinders and incubated at 32°C for 5 h. Aggiutination was monitored
using three methods: 1) top and bottom pH, 2) depth of sediment and 3) total solids.
Top and bottom pH measurements in skim milk were measured up to 5 h with
readings taken at 1 h intervals. The top pH was measured 5 cm below the surface
of the milk. Whereas the bottom pH was measured with the probe resting on the
bottom of the cylinder. The pH differential was computed by subtracting the bottom
pH from the top pH. A pH differential of 0.1 or greater between top and bottom was
used as an indicator of agglutination. The second method to determine
agglutination was depth of sediment, which was measured through the side of the
graduated cylinder using a centimeter ruler. For the final method, samples of the
curd were collected at the end of the 5 hr incubation period and stored at 4°C
overnight for total solid determination the following day. Three gram duplicate
samples were taken from 5 cm below the surface and from the bottom of the
cylinder. Total solids were determined the day after sampling. Samples were dried
in 100°C oven for 16 h, cooled in a desiccator until a constant weight was obtained

for total solid measurements. The % total solids =[(dry weight) x 100] [wet weight

28

(Hicks and Hamzah, 1992). The differential was determined by subtracting the top

from the bottom total solids.

2.5.1 Sample preparation for verification of agglutination in skim milk

2.5.1.1. Effect of heat on the stability of bovine milk immunoglobulins and
their ability to bind lactococci

Insure media was fortified with a 1:5 ratio of antibody to buffered salt solution
and then divided into aliquots of 125 ml. Samples were heat treated for 45 min at
71, 75 or 80°C in a water bath. The media was then cooled to room temperature
under cold tap water and stored at 4°C for 16 h. The media was brought to room
temperature prior to inoculation with the bacterial culture. Next, the cultures were
incubated overnight at room temperature (~ 23°C) until inoculation into the skim milk
as described previously in Section 2.5.
2.5.2.2. Effect of enzyme treatment on the stability of bovine milk

immunoglobulins and their ability to bind lactococci enzyme study
verification

Insure media was fortified with a 1:5 ratio of antibody to buffered salt solution
solution and then divided into aliquots of 125 ml. Papain enzyme concentrations 0
Ulnl, 27.1 Ulnl, 54.3 U/nl, 81.3 Ulnl, 108 U/nl, and 135.6 mm were incubated for 2 h
at 37°C prior to pasteurization at 71°C for 45 min. Next the cultures were incubated

overnight at room temperature (~ 23°C) until inoculation into the skim milk as

described in previously in Section 2.5.

29

2.6. Statistical analysis

The ELISA, milk verification and screening experiments were replicated four
times in a randomized block design. The data was analyzed using a one-way
repeated measures Analysis of Variance with Sigma Stat Analysis System Software
(Jandel Scientific, San Rafael, CA) with the exception of the enzyme ELISA study
which used a nonparametric test based on ranks, for lgM and lgA stability to papain
treatment when binding to M27, and lgM and lgG stability to papain treatment when
binding to M30. Pepsin treatment to lgA for the cultures M27 and SG1 also used a
nonparametric test because the data violated the assumptions of a parametric test.
Statistical differences were computed by the Student-Newman-Keuls method, with a

P<0.05 considered statistically significant.

30

Results and Discussion

3.1. Screening of cultures for agglutination behavior.

3.1.1. Culture agglutination as determined by the ELISA

The agglutination behavior of lactococci determined by the screening method
of Hicks and Ibrahim, (1992) together with the suppliers reported agglutination
behavior are shown in Table 1. The classification of E72 and BGZ as resistant by
the method of Hicks and Ibrahim (1992) was in sharp contradiction to agglutination
behavior reported by the supplier Chris Hansen. Scheuble et al. (1989) stated that
the severity of agglutination for a particular strain depends on two factors: 1) the
frequency with which a specific antigenic determinant is expressed on the cell
surface and 2) the agglutination titer. Thus, the amount of immunoglobulin that
bacteria bind could be used as an indicator of agglutination.

The agglutination behavior of bovine IgM, lgG and lgA to bind lactococci are
presented in Figure 1 and Table 2. The agglutination sensitive single strain cultures
M52, E72 and BG2 showed the greatest affinity (P<0.05) for binding lgM with M52
and E72 binding similar amounts (Table 2). The M30 culture bound less IgM than
the single strain cultures M52, E72 and BG2, yet greater than the other lactococci

WWA, SG1, OS, 478, 874, and M27. The two moderate agglutinating cultures 478

31

and 874 bound Similar amounts of IgM (Table 2). Aggiutination resistant WA, 08

and SG1 along with agglutination sensitive M27 showed the least affinity

Table 1. Comparison of agglutination behavior of lactococci provided by the
supplier, to the screening method of Hicks and Ibrahim (1992).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Culture Aggiutination behavior Hicks and Ibrahim
provided by supplier screening Method
(1992)
WA resistant resistant
OS resistant resistant
SG1 resistant resistant
478 minor to moderate minor to moderate
874 minor to moderate minor to moderate
M52 minor to moderate minor to moderate
E72 sensitive resistant
BGZ sensitive resistant
M30 sensitive sensitive
M27 sensitive sensitive

 

 

to bind IgM (Figure 1; Table 2). With the exceptions of VVWA, 478 and 874 and also
E72 and M52 which bound similar amounts, all other treatments were different
(P<0.05) from each other (Table 2). The agglutination sensitive M27 culture bound
the least amount of IgM for all lactococci tested in my research with the exception of
SG1.

SG1 showed the least affinity for binding lgG (Figure 1; Table 2), whereas
VVWA and OS bound similar amounts of lgG (Table 2) but more than SG1. E72 and

862 showed the greatest affinity to bind lgG (Figure 1; Table 2).

32

Table 2. Ability of single and mixed strain lactococci to bind bovine milk
immunoglobulins as determined by the ELISA.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Culture IgM lgG lgA
Optical Density Mean1
WWA 0.334a 0.590a 0.304a
(0.12) (0.21) (0.10)
OS 0.297" 0.5068 0.242"
(0.11) (0.22) (0.07)
SG1 0.202" 0.363" 0.215C
(0.07) (0.15) (0.08)
478 0329a 0.7650 0.265"
(0.15) (0.23) (009)
874 0.344a 0696d 0.260"
(0.14) (010) (0.09)
M52 0.712d 0.757d 0.662f
(0.26) (0.18) (0.19)
E72 0.745d 0.9869 0.8399
(0.41) (0.18) (0.20)
BG2 0.8856 0947’ 0606"
(0.21) (0.20) (0.11)
M30 0422" 0.754"l 0312°
(0.14) (0.15) (0.12)
M27 02749 0.764if 0.256"
(0.09) (013) (011)

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

33

 

 

 

 

 

 

.0

8 6
0 o

>:mcoo

4 2

o o

23:60

>2mcoo

 

0
0

36:60

.22

on:
NOm
Nnm
mm:
who

wow

wO

.<>>\S

 

8 6
o 0

>:mcoo

 

4 2
o o

36:60

.. NmS.

.. 5N2
.. on:
_. NOD

.. mum

vnm

CuHures
Figure 1. Ability of single and mixed strain lactococci cultures to bind IgM, lgG and

IgA as determined by the ELISA

FOm

mo

34

Both the moderate agglutinating and those sensitive to agglutination showed a
greater affinity to bind lgG than the agglutination resistant cultures WWA, SG1 and
OS (Figure 1; Table 2).

Kanno et al. (1976) determined that IgM was the main antibody causing
agglutination and lgG was a factor due to its large quantities. It was observed with
the ELISA data that the single strain nonagglutinating culture WWA binds relatively
more IgM and lgA than the single strain moderate agglutinating culture 478.
However, 478 binds more lgG than WA. The fact that WA is a nonagglutinating
culture that binds more IgM and lgA than the moderate agglutinating 478 suggest
that lgG may play a larger role in determining culture agglutination behavior than
previously expected. In this study those lactococci that agglutinated tended to bind
more lgG than lactococci that did not agglutinate (Table 2). The ELISA results
(Table 2) suggest that lactococci which bind high quantities of lgG will agglutinate.

M52, E72 and BG2 showed the greatest affinity to bind lgA, whereas SG1
showed the least (Figure 1; Table 2). The M30 and WA cultures bound lgA at a
lower quantity than single strain agglutinating cultures M52, BG2 and E72 (Figure 1;
Table 2). The remaining cultures with different agglutination classifications bound
similar amounts of lgA (Table 2). The ELISA results (Table 2) suggest the binding of
IgA does not play a major factor in the agglutination of cultures since both
nonagglutinating, moderate agglutinating, and agglutinating sensitive cultures bind

similar quantities of lgA.

35

The results of the ELISA (Figure 1; Table 2) indicate that single strain
agglutination sensitive cultures 862 and E72 bind more bovine IgM, IgG and IgA
than the single strain minor agglutinating cultures M52, 478 and 874. The ELISA
results (Figure 1; Table 2) indicate that E72 and BG2 bind large quantities of bovine
immunoglobulins which indicates that these lactococci are sensitive to agglutination,

and thus supports the agglutination behavior reported by the supplier of the cultures
and contradicts the screening method of Hick and Ibrahim (1995). The mixed strain
agglutination cultures M30 and M27 bound more IgM, lgG and lgA than the mixed
strain agglutination resistant cultures OS and SG1 with the exception of OS which
bound more lgG than M27 (Table 2). For the most part the single strain cultures
bound more immunoglobulins than mixed strain cultures with the same agglutination
description (i.e., resistant, moderate, sensitive). This is most likely due to the
presence of agglutination resistant strains within an agglutination sensitive mixed

strain culture.

3.1.2. Aggiutination behavior as determined by laser scanning microscopy
Microscopic analysis of the cultures with LSM using immunofluoresence to
detect lgG confirmed the ELISA results (Table 2). The ELISA results (Table 2)
determined that lactococci bound more lgG than IgM or IgA and for that reason IgG
was Chosen as the fluorescent staining agent. lgG was also previously used in

fluorescent work done by Salih and Sandine, (1984). Aggiutination sensitive

36

 

Figure 2. Laser scanning micrograph of lactococci treated with bovine milk
immunoglobulins: WWA (a,b), E72 (c,d), SG1 (e,), M30 (g,h). a,c,e, and g =
phase contrast; b,d,f, and h = fluorescent images stained with fluorescein
conjugated bovine anti lgG. Magnification 4000x

37

cultures E72 and M30 showed the greatest affinity to bind lgG. This was shown by
the bright spots on the fluorescent images (Figure 2 (d, h)). Phase contrast images
of E72 and M30 showed clumping or chain formation typical of agglutinating cultures
(Figure 2(c, g)). In contrast the agglutination resistant cultures WA and SG1
bound less lgG than the agglutination sensitive cultures (Figure 2 (b, d)). Phase
contrast showed WA and SG1 in smaller chains with few clumps. As with the
single strain cultures, a comparison of the fluorescent images shows that the
agglutinating culture M27 binds more IgG antibodies than the agglutination resistant
culture SG1 (Figure 2 (b, d) and Figure 2 (f, h)). This supports the ELISA data
(Table 1) in which M27 binds twice as much lgG as the nonagglutinating SG1. The
ELISA data Showed that E72 bound more lgG than M27, this was supported by the
results seen from the LSM in which the agglutinating culture E72 binds more lgG
than M30 as evidenced by the bright fluorescence in Figure 2(d, h).

Scheuble et al. (1989) reported no correlation between bacterial chain length
and agglutination. However, Scheuble et al. (1989) believed that strains with a
tendency to form long chains and are sensitive to immunoglobulins will Show an
increase in defect occurrences under commercial production conditions. An
example of this was (Table 2) when WVVA, a nonagglutinating culture, binds similar
amounts of IgM and lgG, in addition to more lgA than many of the minor
agglutinating cultures M27, M30, 478, and 874. This was verified visually in Figure

2 (a, b) in which WWA, even after being exposed to colostrum antibodies still

38

consisted of small chains, whereas E72 and M30 appear as long Chains or clumps
as seen in Figure 2(c, d) and Figure 2(g, h). Due to the small chain size, the weight
of the bacteria casein complex does not reach the critical mass (Milton et al., 1990)
needed for the clumps to sediment to the bottom of the vat.

Over the years, various techniques have been used to determine
agglutination during cheese production based on the physical characteristics or
defects associated with agglutination. The main methods are: 1) sediment depth 2)
microbial enumeration 3) top and bottom total solids 4) direct microscopic
examination. This information allowed dairy processors to choose a corrective
action to reduce agglutination. The main draw back to these four procedures is that
they are performed in the vat while production occurs, thus corrections can only be
made to future incubations with yield loss or batch failure occurring during the test.
Due to the monetary consequences associated with these “in the vat techniques”,
three screening procedures have been developed to determine bacterial sensitivity
to agglutination. However, these procedures require the operator to determine the
endpoints and reaction intensity leaving subjective differences from operator to
operator.

The traditional method for the quantitation of immunoglobulins has been
radial immunodiffusion (Mancini et al., 1965). The drawbacks of this assay are low
sensitivity, time consuming, and requires large volumes of anti-sera. In recent

years, immunoassays have been replacing standard methods of food analysis due

39

to their sensitivity, Specificity and ease of application. Immunoassays offer several
advantages over conventional procedures. They can be very specific and extremely
sensitive, a combination that can result in simpler and quicker protocol procedures
(Rodrigues et al., 1990). The two most common immunoassays in the food area are
the ELISA and radioimmunoassay (RIA). The ELISA is undoubtedly the most widely
used form of immunoassay in food analysis due to its simplicity and suitability as a
screening method. The ELISA is easy to perform, sensitive, specific and relatively
inexpensive (Rodrigues et al. 1990). The main advantage that the ELISA holds
over the RIA for the dairy industry is that the RIA does not require the use of
radioactive isotopes which make the RIA assay difficult for every day industry
application.

Emmons et al. (1963) observed that immunoglobulins are always present in
bulk milk. In addition to this Zawistowski and Mackinnon (1993) showed that up to
89% of Canadian raw milk was contaminated with colostrum. Since antibodies are
present in raw milk, a rapid method to screen cultures for agglutination sensitivity
would be advantageous for the dairy industry. Thunell et al. (1984), suggested that
the selection of starter bacteria from the nonagglutinating strains would be the most
effective method to reduce agglutination. Using an ELISA to screen cultures offers
several advantages over conventional procedures. Ability to screen large numbers
of samples combined with simplicity, sensitivity, and specificity, make the ELISA an

ideal vehicle for use in the dairy industry. In this study, a rapid, objective method to

40

screen large numbers of bacteria was developed. The ELISA technique’s versatility
lends itself to rapidly screening cultures without the expenses associated with an “in
the vat technique.” One main advantage to the ELISA procedure is the elimination of
the subjective determinations made by the operator. The ELISA is an objective
assay that uses Optical densities to determine agglutination, and does not require
extensive training as many procedures including the those developed by Emmons et

al. (1965), Ellerrnan (1975), and Salih and Sandine (1984).

41

3.2 Effect of heat treatment on the stability of bovine milk immunoglobulins

3.2.1. Antibody stability to heat treatment as determined by the ELISA

The effects of heat treatment at 71, 75, 80 and 85°C on the capabilities of
bovine IgM, lgG, and lgA to bind lactococci are presented in Figures 3-5. M30 and
M27 are commercial cultures used for Cheddar cheese production and represent
the extreme case of agglutination problems. The SG1 culture on the other hand was
6 Cottage cheese culture which typically does not agglutinate and was used as a
negative control in this study. At a constant temperature, the percent binding of
immunoglobulins decreased (P<0.05) as heating time increased for all
immunoglobulins (Figures 3—5). This was most notable (P<0.05) with the highest
temperature of 85°C in which percent binding to lactococci was reduced to near zero
levels within the first 20 min of heat treatment. Furthermore, at a constant time, the
percent binding of immunoglobulins to lactococci decreased (P<0.05) with increases
in temperature (Figure 3—5). Percent binding of lactococci was highest (P<0.05) at
the lowest temperature of 71°C and lowest (P<0.05) at the highest temperature of
85°C (Figure 3—5). IgM was still able to bind all three lactococci after 90 min of
heating at 71°C (Figure 3). When heated to 75°C, IgM showed less affinity to bind
the lactococci than when treated at 71°C. The same IgM immunoglobulin when

heated at 80 or 85°C bound less lactococci than those treated to lower temperatures

as evidenced in Figure 3. The data in Table 3 shows that increasing the

temperature from 71 to 75 or 80°C decreased (P<0.05) IgM binding to all three

lactococci. The data (Table 3) shows that the binding ability of IgM to lactococci was

reduced (P<0.5) dramatically at 80°C. Kanno et al. (1976) reported that heating for

10 min at 70°C destroyed almost all of the activity of IgM which they suggested was

the primary agglutination factor. This research showed (Figure 3; Table 3) that

bovine immunoglobulins are still capable of binding lactococci after heat treatments

at higher temperatures and longer times than that recommended by Kanno et al.

(1976)

Table 3. Heat stability of bovine IgM and its ability to bind to lactococci after heat
treatment of 45 minutes.

 

 

 

 

 

 

 

 

 

 

Starter Culture
Heat
Treatment [(430 M27 SG1
(°C)
Optical Density Mean1
71 0.779 a 0.838 a 0.621 a
«110) «108) «118)
75 0.419 " 0.422 " 0.450 "
«114) «120) «117)
80 0.061 ° 0.102 ° 0.189 °
«112) «113) «110)

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

43

 

PercentControl OD PercentControl OD

Percent Control OD

100

80

60

4O

20

100

80

60

40

20

100

80

60

40

20

 

 

M30

>40

 

71C
75C
SOC
85C

 

 

 

LA

l f i
[I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o 20 40 60 80 100
O 71 C
M27 v 75C
3. A 80C
D 85C
3.
o 20 4o 60 80 100
O 71 C
I— A 80C
D 85C
0 20 — 40 60 80 100

Time (Minutes)

Figure 3. Heat stability of bovine IgM and its ability to bind lactococci as determined
by an ELISA Optical density in the unheated sample was 100%.

The bovine immunoglobulin lgG Showed the greatest affinity to bind
lactococci when heated to the lowest temperature of 71°C (Figure 4). When treated
at 75°C, lgG affinity to bind lactococci decreased (P<0.05) as compared to lgG
treated at 71°C (Figure 4; Table 4). The same antibodies when treated at 80 and
85°C, showed little affinity to bind lactococci as evidenced in Figure 4. Raising the
temperature to 75 or 80 from 71°C decreased (P<0.05) lgG binding capabilities to
lactococci (Table 4). My results are consistent with those of De Wit and Klarenbeek
(1984) who reported that 196 was unfolded at 72°C in 0.7 M phosphate buffer, and
also Fukumoto et al. ( 1994) who reported 0 values on bovine lgG of 329 min at
70°C, 38.8 min at 74°C, and 4.3 min at 80°C using radial immunodiffusion.
Dominguez et al. (1997) determined that no loss in antigen binding of lgG occurred
after 60 min at 65°C. My research revealed that the majority of the decreases in lgG
binding occurred when the temperature was raised from 71°C to 75°C (Table 4;
Figure 4). Dominguez et al. (1997) also reported that at 72°C it took only 1387
seconds for a 90% reduction in the binding activity of lgG and suggested that the
ability of lgG to bind antigens after heat treatment has probably been overestimated
in the literature. In my research, 71 or 75°C (Figure 4; Table 4) did not reduce
immunoglobulin binding activity of IgG to the same extent as that reported by
Dominguez et al. (1997). One difference between my research and the work of
Dominguez et al. (1997) was the type of ELISA used where my research detected

only those antibodies that bind lactococci, Dominguez et al. (1997) was interested

45

in all lgG immunoglobulins. Secondly, Dominguez et al. (1997) performed their

heat studies on colostrum, whereas in this research, the heat treatment was

performed on antibodies contained in a whey based media. Li-Chan et al. (1995)

reported slightly higher D values for lgG than Dominguez et. al. (1997). Li-Chan et

al. (1995) performed their research on serum lgG, which is composed of two

subclasses of I96 (55% lgG1 and 45% lng). Whereas, bovine milk lgG is

composed of 90% lgG1 and 10% lng. It is not known whether these subclasses

show different denaturation behavior (Li-Chan et al., 1995). Li-Chan et al. (1995)

concluded that a substantial portion of the antibody activity was retained in milk after

commercial processing.

Table 4. Heat stability of bovine lgG and its ability to bind to lactococci after heat
treatment of 45 minutes.

 

 

 

 

 

 

 

 

 

Starter Culture
Heat
Treatment M30 M27 SG1
(°C)
Optical Density Mean1
71 1.159a 1.851 a 1.235"
(0.30) (0.20) (0.36)
75 0.520 " 0.503 " 0.603 "
(0.07) (0.09) (0.06)
80 0.167" 0.128" 0195°
(0.05) (0.03) (0.10)

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

46

 

6O

4O

20

Percent Control OD

100

80

60

40

Percent Control OD

20

120

100

80

60

40

Percent Control OD

20

 

    

 

 

 

DDCO

71C
75C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time (Minutes)

M30
" 80C
85C
0 20 40 60 80 100
0 71C
M27 V 750
.— A 80C
D 85C
0 20 4O 60 80 100
0 71C
SG1v 75C
.- A 80C
D 85C
r-
blunk“
0 2O 4O 60 80 100

Figure 4. Heat stability of bovine IgG and its ability to bind lactococci as determined
by an ELISA. Optical density in the unheated sample was 100%.

47

Table 5. Heat stability of bovine lgA and its ability to bind to lactococci after heat

treatment of 45 minutes.

 

 

 

 

 

 

 

 

 

Starter Culture
Heat
Treatment M30 M27 SG1
(°C)
Optical Density Mean1
71 0.072 ° 0.065a 0072°
(0.01) (0.01) (0.01)
75 0.055 a 0.049 a 0.053 a
(0.01) (0.01) (0.01)
80 0.022 " 0016° 0034°
(0.03) (003) (0.01)

 

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent
treatments.

Of the bovine immunoglobulins lgA showed the least affinity to bind
lactococci after thermal treatment as Shown by the low optical densities at 71°C
(Table 5). lgA showed the greatest affinity to bind lactococci at the lower
temperatures of 71, and 75°C (Figure 5). When lgA was heated to temperatures
greater than 80°C, the binding activity of lgA decreased (P>0.05) (Figure 5; Table
5). The data (Table 5) showed no statistical differences in lgA binding when the
temperature was raised for commercial cultures M27 and SG1. Raising the

temperature to 80 from 71 or 75°C decreased (P<0.05) IgA antibody binding to M30

(Table 5).

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

120
0 71C
0 M30 v 75C
0 100 — A 80C
0 85C
3 80 -
H
c
o
O 60 -
c
a) 40 b
8
0
CL 20 -
o, _:A;AL 1
0 20 40 6O 80 100
120
0 71C
0 M27 v 75C
0 100 - A soc
_. 0 85C
0
z 80 _
c
o
o 60 '—
E
m 40 -
o
0
CL 20 -:
0, AAiAAL 1
0 20 40 60 80 100
120
0 71C
0 SG1 V 75C
0 100 '- A 80C
_ :1 85C
0
z 80 -
c
O
0 60%
o.-
c
ID 40 3-
o :
L
a)
Q 20 -
0‘ :MAL J_
O 20 4O 60 80 100

Time (Minutes)

Figure 5. Heat stability of bovine lgA and its ability to bind lactococci as determined
by an ELISA. Optical density in the unheated sample was 100%.

49

De Wit and Klarenbeek (1984) reported that reversible changes of protein
structure mostly occur with temperatures up to 60°C. Lindstrom et al. (1994)
concluded that thermal treatment of bovine immunoglobulins will cause the
antibodies to unfold and aggregate. They reported that the unfolding of bovine
antibodies due to heat at 80.9 1: 004°C is irreversible. This was consistent with
Ruegg et al. (1977) who determined that the thermal unfolding of immunoglobulins
was at 81°C in simulated milk ultrafiltrate. De Wit et al. (1983) reported that the
immunoglobulin fraction denatured at 79°C at a 10% level in water. This was
supported by the Figures 3—5 in which after heat treatment at temperatures greater
than 80°C, the antibodies began to lose their binding affinity.

Differences in the results between this research and the literature data might
be attributed to differences in the heating rate. Previous studies performed to
determine the denaturation of immunoglobulins used volumes from 25 pl to 2 ml. My
research used a larger volume of 50 ml needed to encompass the wide range of
samples. This larger volume led to longer heating and cooling times which could
have led to the inactivation of immunoglobulins. Also De Wit and Klarenbeek
(1984), Lindstrom et al. (1994), Ruegg et. al. (1977) and De Wit et al. (1983) used
differential scanning calorimetry (DSC) to determine protein unfolding, whereas my
research focused on the effect of heat played on the binding ability of

immunoglobulins to lactococci.

SO

3.2.2. Antibody stability to heat as determined by laser scanning microscopy

Hicks and Ibrahim (1992) reported that the formation of long chains or clumps
of bacteria are associated with bacterial agglutination. Whereas, smaller Chain
length and no clumping is associated with cultures that do not agglutinate. Some of
the cells appears to be brighter than the rest (Figure 6f). This was explained by the
fact that this lactococci is a mixed strain culture. The brighter fluorescence is due to
the presence of an agglutination sensitive strain contained within an agglutination
resistant culture. Autoclair and Vassel (1963) reported that mixed and multi strain
cultures, although characterized as agglutination resistant may, contain
agglutination sensitive strains which may become dominant upon extensive
subculturing. Furthermore, although no phage problems were experienced in this
research, the existence of phage could compound the agglutination problem if the
agglutination resistant culture was destroyed, leaving the agglutination sensitive
culture to complete the fermentation.

Microscopic analysis of the cultures with LSM using immunofluorescence to
detect lgG, IgM, and lgA confirmed the ELISA results (Figures 6-8). Unheated
immunoglobulins showed the greatest affinity to bind lactococci. This was shown by
the heavy shining Spots on the fluorescent images (Figures 6b, 7b, and 8b). The
phase contrast images showed some clumping or chain formation of cells typical of
agglutinating cultures (Figures 6a, 7a, and 8a). When immunoglobulins were

heated at 71°C for 45 min the lactococci showed similar clumping and chain

51

 

Figure 6. Laser scanning micrograph of lactococci with heat treated lgG: unheated
control (a,b), heated at 71°C for 45 min. (c,d), heated at 75°C for 45 min. (e,f), and
heated at 80°C for 45 min. (g,h). a,c,e, and g = phase contrast; b,d,f, and h =
fluorescence, stained with fluorescein conjugated bovine anti lgG. Magnification

52

length (Figures 6c, 7C, and 8c) as the untreated control (Figures 6a, 7a, and 8a).
The affinity of IgM and lgA to bind lactococci decreased (Figures 7d and 8d)
compared to the unheated control. Whereas, the affinity for lgG (Figure 6d)
remained Similar to the control as seen by the level of brightness (Figure 6b). When
the immunoglobulins were heated at 75°C, the lactococci showed affinity to bind
lgG, IgM, and lgA but to a lesser extent (Figure 6f, 7f, and 8f) than the unheated
control and those heated at 71°C. Phase contrast showed smaller clumps and chain
formation in comparison to unheated control. The immunoglobulins treated at 80°C
showed very little or no binding to lactococci evidenced by very little or no binding of
flourescein conjugated anti immunoglobulins. This is represented by the light to
faint spots on the fluorescence images (Figures 6h, 7h, and 8h). Phase contrast
showed lactococci forming short chains with little or no clumping.

Fukumoto et al. (1994) who reported that lgG was more heat stable than lgA
due to the composition of bovine milk or the structure of lgG. My research supports
the work of F ukumoto et al. (1994) as the binding abilities lgA was reduced when
heat treated (Figures 8 (d, f, h)) as Shown by little to no fluorescence. lgG was still
capable of binding to lactococci after heat treatment at 80°C (Figure 6h) This
correlates with the ELISA data where heat treated lgA retained little binding
capabilities (Figure 8; Table 5). My research was consistent with that of Lindstrom
et al. (1994), Ruegg et al. (1977), De Wit et al. (1983), in that the ELISA and LSM

data Show that the immunoglobulin affinity decreased quickly when heat treated to

53

 

Figure 7. Laser scanning micrograph of lactococci with heat treated IgM: unheated
control (a,b), heated at 71°C for 45 min. (c,d), heated at 75°C for 45 min. (e,f), and
heated at 80°C for 45 min. (g,h). a,c,e, and g = phase contrast; b,d,f, and h =
fluorescence, stained with fluorescein conjugated bovine anti IgM. Magnification

4000x.

54

 

Figure 8. Laser scanning micrograph of lactococci with heat treated lgA: unheated
control (a,b), heated at 71°C for 45 min. (c,d), heated at 75°C for 45 min. (e,f), and
heated at 80°C for 45 min. (g,h). a,c,e, and g = phase contrast; b,d,f, and h =
fluorescence, stained with fluorescein conjugated bovine anti lgA. Magnification

4000x.

55

temperatures above 80°C (Figures 3-5; 6(g, h), 7(g, h), and 8(g, h)).

3.2.3. Verification in skim milk

The rate of top and bottom pH decrease in pasteurized skim milk inoculated
with lactococci grown in media heated at 71, 75, and 80°C is shown in Figure 9.
The SG1 and M27 cultures had faster acid development and a smaller pH
differential (Table 6) than M30 (Figure 9). Skim milk inoculated with either SG1 and
M27 had a similar rate of acid production at the top and bottom of the graduated
cylinder for heat treatments at 71, 75, and 80°C (Figure 9). Table 6 shows the pH
differentials between top and bottom for skim milk at the same heat treatment.
Milton et al. (1990) used a pH differential greater than 0.2 as an indicator of culture
agglutination, whereas Hick and Ibrahim ( 1992) used 0.09 units to indicate
agglutination. In this study a pH differential of 0.1 or greater was selected which
was also used previously by Ibrahim (1995).

Neither SG1 or M27 showed signs of agglutination throughout the 5 hours of
incubation when grown in media heated at 71, 75, and 80°C (Table 6). Visual
inspection of the graduated cylinders at the end of the 5 h incubation Showed no
sediment in all heat treatments of M27 and SG1. Milton et al. (1990) reported that
agglutination of lactococci can occur within the first 15 min of a cheese fermentation.
These effects are also seen in the work reported here (Table 6) where after 2 h

media heated at 71 and 75°C have surpassed the 0.1 pH differential set to

 

  
 
 

  

o to C Top

0 80 'C Bottom
'v 75°C Top
'

CI

-

      
 

75 'C Bottom
71'C Top
71 'C Bottom

    
   
 
    
 

 

pH units

 

 

 

 

 

 

80 "C Top
801C

75 C

75 "C Bottom
71 ’C Top
71 ‘C Bottom

 

 

IDOQOO

 

pH units

 

 

 

 

 

80 °C Top
80 "c Bottom
75 '1C Top
75 _‘C Bottom
71 “'C Top
71 '1C Bottom

IDOGOO

 

 

 

pH units

 

 

 

T Im e (H r s .)
Figure 9. The effect of heat treatment on culture performance measured by pH

differentials. Top and bottom pH decrease in 1000 ml cylinders of skim milk
inoculated with lactococci grown in media heat treated at various temperatures.

57

indicate agglutination, whereas agglutination was not observed in the media heated
to 80°C (Table 6). The agglutination prone M30 culture grown in the media treated
to the higher temperature of 80°C shows less pH differential between top and
bottom for the first 2 h of the incubation. However it was shown (Table 6) that M30
will agglutinate even at the high media pasteurization temperature of 80°C as seen
in hours 3, 4, and 5 (Table 6). After 5 h a pH differential of 0.74, 0.74 and 0.61 was
observed when M30 was grown in media treated to 71, 75 and 80°C, respectively.
Visual inspection of the graduated cylinders incubated with M30 at the end of the 5 h
of incubation had sediment on the bottom of the cylinder. The data suggest that
heat treatment might prevent agglutination in the early stages of a fermentation and
may provide a method to limit agglutination for bacteria that experience light to
moderate agglutination. But if a culture is prone to agglutination, then it will do so
because of immunoglobulins naturally present in the milk which have not received
any additional heat treatment. An example of how heat may provide added
protection against agglutination can be seen with the M27 culture which was
characterized as an agglutination sensitive but Shows little to no visible agglutination
(Figure 9; Table 6). The data in Table 6 show that at the end of the 5 h incubation
the samples treated at lower temperatures had greater agglutination characteristics
than the those treated at 80°C. The trends observed in Table 6 were not statistically

significant most likely due to the variability in pH between replications.

58

Table 6. The pH differentialsa in 1000 ml cylinders of skim milk fermented with
lactococci grown in media heated at various temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

Incubation Time (h)
Heat
Treatment Culture 0 1 2 3 4 5

(°C)
71 SG1 0.00 0.00 0.00 0.01 0.01 0.03
75 SG1 0.00 0.01 0.01 0.01 0.01 0.01
80 SG1 0.00 0.00 0.00 0.00 0.00 0.01
71 M27 0.00 0.01 0.02 0.05 0.04 0.05
75 M27 0.00 0.01 0.02 0.05 0.05 0.04
80 M27 0.00 0.01 0.01 0.02 0.02 0.02
71 M30 0.00 0.05 0.29 0.50 0.80 0.74
75 M30 0.00 0.05 0.17 0.49 0.70 0.74
80 M30 0.00 0.03 0.05 0.36 0.60 0.61

 

 

 

 

 

 

 

 

 

apH differentials = top pH - bottom pH. Results are a mean of 4 independent
treatments. Data within treatments were not statistically different.

M30, M27, and SG1 are multiple strain commercial cultures made from a
defined number strains of lactococci. Although the bacteria were propagated from
the same can of starter culture it is believed that during the individual replication that
one strain or another became the dominant strain. Since each strain has a different
affinity for agglutination, it is believed that this caused the larger variation in the data

evidence by the large standard deviations.

59

The data reported here suggested that heating media and antibodies to
71 °C, although sufficient for pasteurization, was not entirely effective to reduce the
immunogenicity of the milk antibodies that cause agglutination (Figures 3—5). This
study showed that bovine immunoglobulins are still capable of binding lactococci
after heat treatments at higher temperatures and longer times than that
recommended by Emmons et al. (1966); Kanno et al. (1976) and Dominguez et al.
(1997). An increase in heat treatment to 75°C or 80°C may prevent agglutination in

the bulk media.

60

3.3. Enzyme Treatment of immunoglobulins to reduce agglutination.

3.3.1. Antibody stability to enzyme treatment as determined by the ELISA

The effect of papain treatment on the capabilities of bovine IgM, lgG and lgA
to bind lactococci are presented in Figure 10. The binding affinity of IgM was
highest (P<0.05) at the lowest concentration 27.1 U/nl of papain and lowest
(P<0.05) at the higher concentrations of 108 U/nl and 135.6 Ulnl (Figure 10). This
trend was also similar for IgG, however lgA showed the largest (P<0.05) binding at 0
U/nl and than less binding as papain concentration increased. The binding ability of
IgM and lgG increased (P<0.05) (Table 10; Figure 10) after treatment at the lowest
papain concentration of 27.1 Ulnl (Figure 10). This was consistent with the theory
that brief exposure of a protein to papain will cleave the most susceptible bonds,
whereas prolonged exposure will cause the protein to become totally digested
(Kuby, 1992). Brief exposure to papain will cleave immunoglobulins into monmers,
as well as at the hinge region of the "Y" leaving 2(Fab) fragments and a tail section
(Kuby, 1992). The monomers and Fab fragments with intact antigen receptor site
were still capable of binding bacteria, and account for the increased binding of
immunoglobulins (Figure 10). The decrease in the positional hindrances led to
greater antibody binding (Figure 10; Table 7). However, as larger concentrations of

papain were used more of the Fab fragments were digested leaving less antibodies

61

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

350
0 IgM 1:: 861
0 300 — 3 Ni;
’6
Z 250 -
C
O
o 200 ..
E
0 150 -
8
Q)
o. 100 r
50 L L l l 1
27.1 54.3 81.3 108 135.6
350
o t: SG1
O 300 _ IgG 3 :2;
2 250 L
C
o 200 -
o
E 150 -
a)
3 100 -
0
(1 50 ..
o A I
27.1 54.3 81.3 108 135.6
110
SG1
O 1003' 19A 2 M27
M30
0 so 3" °
2 80 -
C
o 70 -
0 60 _
C
a, 50 -
S 40 r-
(D
0 30 l-
20 l 1 1 1 1

 

 

27.1 54.3 81.3 108 135.6
Papain Concentration units/nl

Figure 10. Effect of papain treatment for 2 h at 37°C on the stability of bovine IgM,
IgG and lgA and their ability to bind lactococci as determined by an ELISA Optical
density in the nonenzyme treated sample was 100%.

62

Table 7. The stability of bovine IgM and its ability to bind lactococci after papain
treatment at 37°C for 60 minutes.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Starter Culture
Papain
Concentration M30 M27 SG1
(units/nl)
Optical Density Mean1
0 0.501 a 0395° 0.221°
(0.12) (0.11) (0.14)
27.1 0.780" 0.664" 0.638"
(0.08) (011) (0.16)
54.3 0.581 ° 0.480° 0.477"
(0.07) (0.08) (0.12)
81.3 04923 0887a 0.352d
(0.07) (0.05) 0.08)
108.0 0437a 03598 0.818d
(0.06) (0.05) (0.07)
135.6 0.374a 0.350a 0.322d
(004) (0.06) (0.08)

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent
treatments.

63

Table 8. The stability of bovine IgG and its ability to bind lactococci after papain
treatment at 37°C for 60 minutes.

 

 

 

 

 

 

 

 

 

 

 

 

 

Starter Culture
Papain
Concentration M30 M27 SG1
(units/nl)
Optical Density Mean1
0 0503° 0642a 02963
(019) (0.01) (0.10)
27.1 0.833" 0.761T 0.818"
(0.07) (0.15) (0.09)
54.3 0.673" 0.6053 0.604c
(0.06) (0.09) (0.11)
81.3 0.5188 0.4740 0.436d
(0.06) (0.07) (0.10)
108.0 0.3083 0.304d 03143
(0.08) (0.07) (0.09)
135.6 0.27081 0228‘5 0.236d
(0.04) (0.06) (0.06)

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

64

 

Table 9. The stability of bovine lgA and its ability to bind lactococci after papain
treatment at 37°C for 60 minutes.

 

Starter Culture

 

 

 

 

 

 

 

 

 

 

 

 

Papain
Concentration ((430 M27 SG1
(units/nl)
Optical Density Mean1
0 0.672" 0.5513 o.504°
(0.14) (0.13) (006)
27.1 0.455" 0.4583 0.4833
(0.06) (0.05) (0.03)
54.3 0.316" 0.312" 0.320"
(0.06L (0.04) (0.03)
81.3 0.236d 0.230c 0.2180
(0.03) (0.03) (0.02)
108.0 0179d 0.175d 0169d
40.03) (0.02) (0.02)
135.6 0.153d 0.157d 0.161d
(0.01) (0.01) (0.02)

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

65

 

capable of binding lactococci. The data (Table 7; Figure 10) Shows that the binding
ability of IgM decreased (P< 0.05) as the papain concentration increased for the
cultures M30, M27 and SG1.

The nonagglutinating culture SG1 bound less antibodies than M27 and
M30 at the 0 U/nl concentration level. However after treatment at 27.1 Ulnl, SG1
bound relatively similar amounts Of immunoglobulins as the agglutinating
cultures M30 and M27. Furthermore, SGI bound a relatively greater percent
when compared to the control than either M30 or M27. This may also be an
indication that IgM is the major agglutinating factor (Kanno et al., 1976) because
when the antibody is digested it eliminates the sterohindrances and thus SG1
binds relatively more IgM as a percent of the control than either of the
agglutinating cultures M30 or M27. The results have shown a similar trend for lgG
but a steeper decrease in antibody binding affinity when compared to IgM. As with
IgM, SG1 bound the least amount of lgG antibodies at the 0 Ulnl concentration
level, but upon treatment at 27.1 U/nl, SG1 bound equal amounts of lgG as the
agglutinating cultures M30 and M27. The data from the stability of bovine lgG
and its ability to bind M30 (Table 8) did not pass the assumptions of a
parametric test, therefore a nonparametric test based on ranks was performed
leaving data which appears different, actually similar.

The effect of papain treatment on the binding ability of IgA to lactococci is

shown in Table 9. The binding ability of lgA decreased (P< 0.05) as the papain

66

concentration increased for the cultures M30, M27 and SG1 (Table 9; Figure 10).
The data in this table do not show the initial increase (P<0.05) in the binding ability
of lgA at (27.1 U/nl) as IgM and lgG did. However as with IgM (Table 7) and lgG
(Table 8) the binding capabilities of the antibody decreased when exposed to
higher concentrations of papain (Table 9; Figure 10).

Ustunol and Hicks (1994) reported that papain digestion of antibodies
decreased agglutination not only in the media but also in 1000 ml cylinders of skim
milk. The initial increases in antibody binding shown in this research support the
data reported by Ustunol and Hicks ( 1994) (Tables 7-9). Ustunol and Hicks (1994)
reported that the hydrolyzed fragments specifically those less than molecular weight
of 10,000 daltons bind the antigenic site on the cell surface in the same capacity as
they did when they were part of the intact immunoglobulin. However, with the
bridging mechanism removed the protein can no longer join bacteria to form chains
or clumps. The blockers having effectively removed the antigenic binding Site on the
bacteria are then carried with the bacteria to the milk were they prevent milk
antibodies from agglutinating the bacteria.

The effect of pepsin treatment on the binding capabilities of IgM, lgG and lgA
to bind lactococci can be found in the Appendix (Tables 10-12; Figure 13). These
results were inconclusive. Ustunol and Hicks ( 1994) also reported that proteolytic
enzymes other than papain (such as trypsin, chymotrypsin, and pepsin) liberated

peptides that do not inhibit agglutination. The data in Tables 10-12 had all

67

treatments similar for M30 and M27. This might be explained by the fact that the
enzyme pepsin cleaves antibodies in the bond just above the interchain disulfide
bonds. On an lgG molecule, pepsin produces one main fragment called F(ab)2
fragment and many small fragments that compose the Fc. The F (ab); fragment
consists of the two Fab fragments joined by an interchain disulfide bond which can
precipitate antigens (Kuby, 1992). This was shown in Figure 13 in which as the
amount of pepsin increases, the percent binding of IgM and lgG remains statistically

Similar for M30, M27 and actually increases for the culture SG1.

3.3.2. Antibody stability to enzyme treatment as determined by Laser
scanning microscopy

Microscopic analysis of lactococci with LSM using immunofluoresence to
detect lgG confirmed the ELISA results (Figure 10; Table 7-9). Phase contrast
images showed some clumping or chain formation typical of agglutinating cultures
when treated with papain concentrations of 27.1 Ulnl (Figure 11 (a, e)). The heavy
binding of immunglobulin was evidenced by the large clumps of bacteria and the
heavy fluorescence around the bacteria and clumps (Figure 11 (D, f)). When the
immunoglobulins were treated at a concentration of 135.6 Ulnl the phase contrast
images of the lactococci Showed less clumping and chain binding (Figure 11(c,
g)). The fluorescent images at the higher papain concentration contain heavy

spots of fluorescence for the agglutination resistant culture SG1 (Figure 11d).

68

 

Figure 11. Laser scanning micrograph of commercial culture SG1 grown in

Insure media after papain treatment at: 27.1 U/nl (a,b), 135.6 Ulnl (c,d), culture M30
grown in Insure media after papain treatment at 27.1 U/nl (e,f), 135.6 U/nl (g,h).
a,c,e, and g = phase contrast; b,d,f, and h = flu0rescent images stained with
fluorescein conjugated bovine anti lgG. Magnification 4000x.

69

Whereas the agglutination sensitive culture M30 appears as faint spots on the
fluorescent images indicating little immunoglobulins are still present (Figure
11h). LSM results for the pepsin treatment are provided in the Appendix. As with
the ELISA data there were no identifiable reductions in agglutination for either the
phase contrast images (Figure 15 (a, c) and 15 (g, h)) or the fluorescent images
(Figure 15 (b, d) and 15 (f, h)) after pepsin treatment of immunoglobulins.

In the LSM photomicrographs for papain (Figure 11), the transmitted image
shows a decrease in agglutination between the 27.1 Ulnl treatment and the higher
papain concentration 135.6 Ulnl treatment. At the higher papain concentrations
(Figure 11 (C, 9)) the decrease is Characterized by smaller chains or clumps of
bacteria. This supports the research of Ustunol and Hicks ( 1994) who saw a
decrease in chain length and clumping of bacteria when grown in media treated with
papain. When immunoglobulins were treated at the higher papain treatment of
135.6 Ulnl their was a noticeable decrease in fluorescence for the agglutinating
culture M30 (Figure 11(f, h)) . Less fluorescence means that fewer antibodies are
still capable of binding the surface cell receptors of the bacteria resulting in less
agglutination. This supports the theory that prolonged exposure of antibodies to
papain will digest the entire antibody. These pictures (Figures 11(f, h)) support that

theory.

70

3.3.3. Verification in skim milk

From the results of the ELISA and LSM, papain concentrations 27.1 U/nl,
81.3 U/nl and 135.6 Ulnl were chosen for reduction of agglutination in a milk study.
Unfortunately the enzyme was not denatured by the pasteurization step and quickly
coagulated the milk leaving the milk study results inconclusive. Ustunol and Hicks,
(1994) used diafiltration to isolate antibody fragments with a molecular weight less
than 10,000 daltons. Commercial cultures OS, M30, and M37 when grown in media
reconstituted with these hydrolyzed fragments showed reductions in agglutination of
40, 55 and 72% respectively. Diafiltration does represent a method to collect the
hydrolyzed proteins from papain digestion while separating out the enzyme (Ustunol
and Hicks, 1994). However attempts to use diafiltration to remove the enzyme in

this milk study was unsuccessful due to equipment limitations.

71

Conclusions

Screening Study

The ELISA can be used to routinely screen large numbers of samples both
quickly and objectively. The ELISA provided a rapid method to determine antibody
binding capabilities, and thus, the agglutination behavior of lactococci. The ELISA
results suggested that lgA plays a minor role in agglutination, whereas the role of

lgG in agglutination may have been understated in the literature to date.

Heat Study

Heat treatment represented a method to denature antibodies as seen by both
the ELISA and laser scanning microscopy. At a constant temperature, the percent
binding of immunoglobulins to lactococci decreased as time of treatment increased.
Additionally, decreases in the percent binding of immunoglobulins to lactococci was
observed with increased temperature at a constant time. The data reported here
suggested that heating media and antibodies to 71° C, although sufficient for
pasteurization, was not effective to reduce the immunogenicity of the milk antibodies
that cause agglutination. An increase in heat treatment to 75°C or 80°C caused
decreases (P<0.05) in IgM, lgG and IgA binding to lactococci. Thus, increasing

heat treatment could help reduce agglutination in bulk milk.

72

Enzyme Study

Papain treatment of immunoglobulins represented a method to denature
antibodies as seen by the ELISA and laser scanning microscopy. However, the
peptides released from the denaturation of antibodies with pepsin did not prevent
agglutination. Upon partial digestion with papain all three antibodies were still able
to bind lactococci. Additionally, initial increases in binding to M30, M27 and SG1

were observed for IgM and lgG but not lgA which showed decreases in antibody

binding.

73

Recommendations

Continued investigation of the heat and enzyme studies with single strain
cultures, as well as the evaluation of other enzymes such as chymopapain, trypsin,
and chymotrypsin for their effect on antibody stability would be a benefit to the
literature. The use of laser scanning microscopy should be investigated with
Splitscreen imaging and multiple antibodies to illustrate the relationship of
agglutination between antibodies. Further investigation into the use of ultra violet
light to kill bacteria, or Chemicals such as formalin reduce the time needed for ELISA

coating would be beneficial in decreasing assay preparation time.

74

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75

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APPENDIX

81

Appendix

Pepsin ELISA and laser scanning microscopy data

Table 10. The stability of bovine IgM and its ability to bind lactococci after pepsin
treatment at 37°C for 60 minutes.

 

 

 

 

 

 

 

 

 

 

 

 

Starter Culture
Pepgn
Concentration M30 [0127 331
(units/nl)
Optical Density Mean
0 0.4523 0.458a 0.255a
(0.11) (0.05) (0.11)
375 0394° 0.450" 0.2188
(0.15) (009) (008)
750 0.343° o.407° 0187°"
(0.12) (008) (004)
1.100 0.3338 0.427a 02181
(0.09) (0.08) (0.08)
1 .500 03953 0432a 0.280ac
(0.08) (0.10) (0.08)
1 .880 0.380111 o.444° 0286"“C
(0.13) (0.10) (0.07)

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

82

 

Table 11. The stability of bovine lgG and its ability to bind lactococci after pepsin
treatment at 37°C for 60 minutes.

 

 

 

 

 

 

 

 

 

 

 

 

Starter Culture
Pepsin
Concentration M30 M27 SG1
(units/nl)
Optical Density Mean
0 0640a 06993 0.4o1°
(0.10) (0.07) (0.17)
375 0.71811 0.6898 0.478a
(.117) (0.07) (0.17)
750 0.651 a 07393 0.454‘1
(0.07) (0.02) (0.10)
1,100 0,7411 0.7513 0.607°"
(0.04) (0.06) (0.13)
1,500 0,7948 0.8168 0.554°"
(0.05) (0.04) (0.17)
1 ,880 0784a 0.6888 0.747"
(0.05) (0.22) (0.06)

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

83

 

Table 12. The stability of bovine lgA and its ability to bind lactococci after pepsin
treatment at 37°C for 60 minutes.

 

 

 

 

 

 

 

 

 

 

 

 

Starter Culture
Pepsin
Concentration M30 M27 SG1
(units/nl)
Optical Density Mean
0 0.4903 0.426a 0.38523
(0.25) (0.22) (0.24)
375 0.27811 0.228a 0.16911
(0.12) (009) (0.11)
750 0258° 0.184° 0.163°
(0.09) (0.06) (0.09)
1,100 0.255a 0.179a 0.167a
(0.08) (0.47) (0.08)
1,500 0.2483 0.181" 0.170a
(0.08) (0.49) (0.06)
1,880 0.2501 0.1778 0159°
(0.08) (0.05) (0.06)

 

 

 

 

1 Optical Density Mean with standard deviation in parentheses.

Means with different superscript are statistically different (P < 0.05) for
comparisons made within the same column. Results of 4 independent

treatments.

84

 

100

90

PercentControl OD

80

70

220

200

180

160

140

Percent Control 00

100

80

64
62

OD

60
58
56
54
52

Percent Control

50

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

D SG1
19M A M27
r- O M30/’0
F
375 750 1100 1500 1875
D 361
.. '96 A M27
0 M30
P
375 750 1100 1500 1875
D SG1
F' lgA A M27
W0
+

 

 

375 750

1100 1500 1875

Pepsin Concentration Units/nl

Figure 12. Effect of pepsin treatment for 2 h at 37°C on the stability of bovine IgM,
lgG and lgA and their ability to bind lactococci as determined by an ELISA. Optical
density in the nonenzyme treated sample was 100%.

85

 

 

Figure 13. Laser scanning micrograph of commercial culture SG1 grown in

Insure media after pepsin treatment at: 375 Ulnl (a,b), 1,880 Ulnl (c,d), and culture
M30 grown in Insure media after papain treatment at 375 U/nl (e,f), 1,880 U/nl (g,h).
a,c,e, and g = phase contrast; b,d,f, and h = fluorescent images stained with
fluorescein conjugated bovine anti lgG. Magnification 4000x.

86

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