Inhibition of clostridia with lactic acid bacteria

The invention relates to inhibition of clostridia with lactic acid bacteria. In particular, the invention relates to the use of Lactobacillus rhamnosus in the food industry to inhibit the growth and activity of clostridia. Preferred embodiments of the invention include the use of Lactobacillus rhamnosus in prevention of butyric acid fermentation and in cheese production.

FIELD OF THE INVENTION 
The present invention relates to inhibition of clostridia with lactic acid 
bacteria. In particular, the invention relates to the use of Lactobacillus 
rhamnosus in the food industry to inhibit the growth and activity of 
clostridia. 
BACKGROUND OF THE INVENTION 
Clostridia are gram-positive, anaerobic, sporeforming bacteria with a 
fermentative metabolism. Clostridia are very effective producers of gas 
(H.sub.2 and CO.sub.2); their other fermentation products include butyric 
acid, acetic acid, butanol, ethanol, isopropanol, acetone and other 
organic acids and alcohols. Clostridia are also the principal agents of 
the anaerobic decomposition of proteins. The soil and water are the 
primary environment of clostridia, although they also may be present in 
poor-quality silage, feeds, fertilizers and contaminated foods, such as 
vegetables, fish, and crustaceans, meat, milk, and sweets. 
Certain clostridia have been found to be pathogenic. Pathogenic clostridia 
are also normally found in the soil. They have little invasive power, but 
instead they produce a variety of highly toxic proteins known as 
exotoxins, and clostridial diseases are a result of such exotoxins. 
Examples of diseases caused by clostridia include botulism and other less 
hazardous food poisonings, which are specifically a result of the 
exotoxins formed in the food. Other severe clostridial diseases include 
tetanus and gas gangrene, resulting from wound infections. Some 
clostridial toxins, such as those responsible for botulism and tetanus, 
are potent inhibitors for nerve function. Others again, such as those 
causing gas gangrene, are enzymes. Such enzymes include lecithinase, 
hemolysin and a variety of different proteases. 
In addition to being pathogenic, clostridia are also otherwise harmful. The 
shelf-life and utility of foods and their raw materials is often impaired 
by clostridial spoilage. Clostridia spoil not only food consumed fresh or 
employed as raw material in the food industry but also products of the 
food industry, such as products of the canning industry, processed meat, 
milk products, etc. The effects of clostridia on food have been described 
in W. C. Frazier, Food Microbiology, 2nd edition, 1967, McGraw-Hill Book 
Company, New York. 
Clostridia may also present problems in food production. Particularly in 
the dairy industry, the adverse effects of food-borne clostridia are 
great. Hence one of the greatest problems attending cheese production is 
false fermentation caused by clostridia during the curing of cheese. 
Clostridia use for example available carbohydrates and lactic acid from 
normal lactic acid fermentation, producing therefrom butyric acid and 
gases, particularly H.sub.2 and CO.sub.2. Such butyric acid fermentation 
will completely spoil the cheese. On account of excessive gas formation, 
the texture of the cheese changes, the cheese swells too much and may even 
explode. The butyric acid formed in cheese has a very strong and 
unpleasant taste, and thus the cheese is not accepted for vending on 
account of the foul off-taste. 
Various means are employed in an attempt to inhibit the growth of 
clostridia. Nitrate and nitrite in the form of various alkali salts are 
used to inhibit clostridial growth for example in meat and meat products 
and in certain cheese varieties. However, nitrates and nitrites are toxic 
at high concentrations, and their use as additives is not desired. In view 
of the consumer attitudes that are increasingly against the use of 
additives and the different food legislations in different countries, 
industry in general currently strives to find natural biopreservatives to 
replace chemical additives. 
Heat treatment, such as sterilization of foods or their raw materials, has 
also been employed in an attempt to inhibit clostridial growth. Spores 
formed by clostridia are nevertheless thermostable, and in the worst case 
heat treatment can even have the opposite effect. In the dairy industry, 
problems are also presented by the fact that pasteurization of milk kills 
other acid-forming bacteria, and thus clostridia which do not normally 
grow very well at low temperatures and on the other hand do not compete 
well with other acidforming bacteria at higher temperatures have no 
competing organisms after the spores have germinated. 
To avoid problems caused by clostridia, it is attempted to keep the 
clostridial spore content in milk as low as possible by completely 
prohibiting the use of silage or by using silage of very high quality 
only. Clostridial spores can also be bactofugated from milk prior to the 
use of the milk as such or as a raw material in the dairy industry. 
Bactofugation, i.e. removal of bacteria and/or spores by centrifugation, 
nevertheless has various effects on the quality of the final product and 
is also uneconomic. 
To inhibit the growth of clostridia, the enzyme lysozyme has also been used 
in several branches of the food industry. The activity of lysozyme is not 
confined to harmful microbes only, but lysozyme also inhibits the activity 
and growth of any desired bacteria. Thus such a treatment also affects the 
quality of the final products and is not recommended for use. 
Lactic acid bacteria are known to produce various antimicrobial compounds, 
such as organic acids, hydrogen peroxide, diacetyl and bacteriocins, and 
it has been attempted to use these as additives for instance to improve 
the shelf-life of food. Commercially available is the product Nisaplin 
(Aplin & Barrett), incorporating purified nisin produced by the bacterium 
Lactococcus lactis and exerting an effect against gram-positive bacteria. 
The use of nisin to control the growth of Clostridium botulinum spores in 
high moisture content cheese spreads is described in U.S. Pat. No. 4 584 
199. U.S. Pat. No. 4 790 994 discloses the use of Pediococcus pentosaceus 
to inhibit the growth of psychrotrophic bacteria in milk products of e.g. 
the cottage cheese type. There is no reference to clostridia in this 
patent. 
WO Patent publication 93/09676 relates to a method for preserving a food 
product by treating the product with an euhygienic bacterial strain 
competitively inhibiting the growth of pathogenic and spoilage organisms 
prior to packaging of the product. Euhygienic bacteria are defined as 
non-pathogenic and/or non-spoilage bacteria. Lactobacillus delbruckii and 
Hafnia alvei are stated as preferred. However, the effect of these species 
has not been shown in the publication. 
European published application 344 786 (A2) discloses a bacterial 
preparation incorporating a Lactobacillus gasseri, Lactobacillus 
delbruckii and/or Bifidobacterium longum culture in a suitable medium, 
such as soy milk, which according to the publication can be used to 
inhibit clostridial growth. The publication indicates that the preparation 
can be used both therapeutically, to inhibit intestinal clostridial 
growth, and as a food preservative, in which case the preparation is used 
to increase the acidity of the product. However, the anticlostridial 
effect of said bacteria has not been demonstrated. 
DE Offenlegungsschrift 31 25 797 (A1) discloses a dietetic preparation 
comprising Lactobacillus casei in a suitable culture medium, such as soy 
milk, which in accordance with the publication can be used to inhibit 
intestinal clostridial growth. The publication indicates that the 
preparation can, like other similar lactic acid bacteria preparations, 
also be used for prophylaxis for example in undergoing antibiotic therapy. 
However, the publication provides no test results of the anticlostridial 
effects of the dietetic preparation or the Lactobacillus casei culture 
incorporated therein, and there is no proof of the effectiveness of the 
invention. Neither is any other utility for Lactobacillus casei described 
or suggested in the publication. 
BRIEF DESCRIPTION OF THE INVENTION 
It was an object of the present invention to find novel solutions to 
problems caused by clostridia. In that connection, special attention was 
paid to the problems presented by clostridia in the food industry. In 
particular, it was attempted to find a solution to the question how 
butyric acid fermentation could be prevented. 
When the effect of the antimicrobial factors of various lactic acid 
bacteria and other bacteria on the growth of clostridia was studied, it 
was unexpectedly found that Lactobacillus rhamnosus has an excellent 
anticlostridial effect. The microorganism Lactobacillus rhamnosus LC-705, 
DSM 7061, was particularly advantageous in its anticlostridial effect. 
Thus the present invention relates to the use of Lactobacillus rhamnosus in 
the food industry to inhibit the growth and activity of clostridia. 
A particularly preferred embodiment of the invention involves the use of 
the strain Lactobacillus rhamnosus LC-705, DSM 7061, in the food industry 
to inhibit the growth and activity of clostridia. 
Lactobacillus rhamnosus LC-705 was deposited on May 13, 1992, as DSM 7061, 
at DSM DEUTSCHE SAMMLUNG VOON MIKROORGANISMEN UND ZELLKULTUREN GmbH, 
Mascheroder Weg 1B, D-3300 Braunschweig and was accorded Accession Number 
DSM 7061. 
The present invention also relates to a method for inhibiting the growth 
and activity of clostridia in a food product, the method being 
characterized in that Lactobacillus rhamnosus is added to the food product 
for inhibition of clostridia. The method of the invention preferably 
employs Lactobacillus rhamnosus LC-705, DSM 7061. 
The present invention further relates to a method for preventing butyric 
acid fermentation, the method being characterized in that Lactobacillus 
rhamnosus is used to inhibit the activity of clostridia. The method of the 
invention preferably employs Lactobacillus rhamnosus LC-705, DSM 7061. 
DETAILED DISCLOSURE OF THE INVENTION 
The present invention, therefore, relates to the use of Lactobacillus 
rhamnosus in the food industry to inhibit the growth and activity of 
clostridia and to prevent butyric acid fermentation. 
The concept of food industry should be broadly construed in this context, 
since it is considered to mean besides the conventional food industry, 
such as the dairy industry, canning industry, meat industry and sweets 
industry, also the feeds industry, for example, i.e. all branches of 
industry the products of which are ingestible by humans or animals. In 
addition to actual industrial products, in the present context this 
concept also refers to raw materials used in these industries and to foods 
consumed fresh, such as vegetables, milk, etc. 
The basis underlying the invention and the practical implementation thereof 
will be described in the following, using as an exemplary organism 
Lactobacillus rhamnosus LC-705, available at the microorganism collection 
of Valio Oy by product number LC705. The strain has been deposited on May 
13, 1992 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH 
(DSM) by number DSM 7061, and has the following characteristics: 
gram-positive short chain-forming rod 
homofermentative 
good growth at 15-45.degree. C. 
no proteolytic activity 
does not produce ammonia from arginine 
catalase-negative 
when grown in an MRS broth (LAB M), the strain produces 1.6% lactic acid 
having an optical activity of the L(+) configuration; the strain 
decomposes citrate (0.169%), producing diacetyl and acetoin; the strain 
ferments at least the following carbohydrates (sugars, sugar alcohols): 
ribose, galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, 
mannitol, sorbitol, methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, 
arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, 
trehalose, melezitose, gentiobiose, D-turanose and D-tagatose 
survives well a salinity of 5% and fairly well a salinity of 10%. 
Lactobacillus rhamnosus has been described in detail in Finnish Patent 
Application 922699, which relates to that strain and its use for 
inhibition of yeasts and moulds. There is no suggestion of the use of this 
strain for inhibition of clostridia in the literature in this field. 
In addition to the above preferred strain, other Lactobacillus rhamnosus 
strains suitable for the purposes of the invention and having an 
anticlostridial effect may naturally also be employed. An example of these 
is Lactobacillus rhamnosus LC-10, available at the microorganism 
collection of Vallo Oy by product number 706. Also other bacterial strains 
having utility in accordance with the invention will be found at 
depositary institutions for microorganisms and in their published 
catalogues. After the disclosure of the present invention, it is obvious 
to those skilled in the art to test for the anticlostridial effect of 
other bacterial strains by simple screening. 
In accordance with the invention, a Lactobacillus rhamnosus strain, 
preferably the strain Lactobacillus rhamnosus LC-705, is employed for 
inhibition of clostridia in the food industry. Depending on the situation, 
the above strain is added either to the fresh food, to foodstuffs used as 
raw materials in the food industry, or in conjunction with the food 
production process. 
The Lactobacillus rhamnosus strain can be prepared for use as a fresh 
culture, diluted from a stock culture, or used in the form of a 
lyophilizate reconstituted either prior to use or upon use. The fresh 
culture can be prepared in the conventional manner, for example by 
culturing the strain in a whey-based medium for 2 to 3 days at a 
temperature of 30-37.degree. C. with or without pH adjustment. For 
example, the preparation of Lactobacillus rhamnosus LC-705 has been 
described in detail in the above Finnish application 922699, incorporated 
herein by reference. The cell-containing fermentation broth is recovered 
and can be used as such, concentrated, or lyophilized. The lyophilization 
is performed by conventional methods. The concentration may be carried out 
with microfiltration apparatus or by other corresponding methods. 
If desired, other bacteria or additives for inhibition of clostridia may be 
added to the preparation incorporating Lactobacillus rhamnosus cells, 
prepared as described above. When bacteria are employed, such another 
bacterial strain can be cultured either simultaneously with or separately 
from the Lactobacillus rhamnosus strain. Simultaneous culturing of the 
strains is of advantage on account of its simplicity and ease. On the 
other hand, separate culturing of the strains makes it possible to vary 
the proportions of the strains to be included in the bacterial 
preparation, if desired. It is naturally also possible to use lyophilized 
preparations of several bacteria, which are reconstituted together or 
separately. 
Even when the Lactobacillus rhamnosus preparation employed contains no 
other substances, it is naturally possible to employ other substances as 
well, such as other substances employed in food processes and other 
anticlostridial agents, in connection with use of Lactobacillus rhamnosus 
in accordance with the invention. For example in the dairy industry, the 
Lactobacillus rhamnosus strain used in accordance with the invention may 
be employed as one starter organism to replace other normal starter 
organisms in the particular process or in addition to them. 
The Lactobacillus rhamnosus strain is used in a quantity sufficient to 
achieve the desired effect, i.e. inhibition of clostridia. The quantity 
employed naturally varies with application. The suitable amount can be 
easily determined for each implementation. 
An anticlostridial Lactobacillus rhamnosus strain, preferably Lactobacillus 
rhamnosus LC-705, can be employed to prevent problems caused by clostridia 
in a number of different fields, for instance to prevent clostridial 
spoilage in products of the food industry and fodder industry, and to 
inhibit the growth of clostridia and prevent clostridia-related false 
fermentation during preparation processes of food and fodder. In addition 
to the use disclosed herein for Lactobacillus rhamnosus, it is naturally 
also possible to employ strains of Lactobacillus rhamnosus for medical 
purposes. 
The invention will be described in greater detail by means of the following 
examples. The examples are only intended to illustrate the invention and 
are in no way to be construed as restricting its scope.

EXAMPLE 1 
Inhibition of clostridia with Lactobaclllus rhamnosus LC-705 strain 
The test series was conducted with milk prepared from 10% milk powder (a 50 
ml). The milk samples were inoculated with a spore suspension of the 
strain Clostridium tyrobutyricum K8 isolated from cheese and the 
Lactobacillus rhamnosus LC-705 strain. Clostridia were added to the milk 
to give an initial clostridial content of about 1000 CFU/ml. Lactobacilli 
were added from a culture grown in a 1% whey broth for 48 hours. The 
samples containing clostridium and lactobacillus inoculum were run through 
a program simulating a cheesemaking process, whereafter the samples were 
transferred to +20.degree. C. The clostridia contents were determined 
either on Reinforced Clostridial Agar (R.C.M., Lab 23) by the plating 
method or in Tyrobutyricum Broth (Merck 11734) by the Most Probable Number 
(MPN) technique. A sample incorporating Clostridium tyrobutyricum K8 only 
was employed as a control. The results are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Anticlostridial effect of Lactobacillus rhamnosus LC-705 strain 
Clostridium tyrobutyricum K8 contents, CFU/ml 
0 h 2 days 
14 days 
23 days 
39 days 
60 days 
__________________________________________________________________________ 
LC-705 
9.8 .times. 10.sup.3 
4.9 .times. 10.sup.3 
&lt;10 &lt;10 &lt;10 &lt;10 
1% 
Control 
8.6 .times. 10.sup.3 
1.1 .times. 10.sup.3 
1.8 .times. 10.sup.4 
2.4 .times. 10.sup.5 
2.3 .times. 10.sup.4 
8.1 .times. 10.sup.4 
K-K8 
__________________________________________________________________________ 
The results show that Lactobacillus rhamnosus LC-705 exhibits an excellent 
anticlostridial effect. 
EXAMPLE 2 
Inhibition of clostridia with different Lactobacillus strains 
The tests described in Example 1 were repeated employing the following 
strains of lactobacilli: 
1. Lactobacillus rhamnosus LC 705 (DSM 7061) 
2. Lactobacillus rhamnosus LC-10 (Valio 706) 
3. Lactobacillus plantarum NRRL-B-193 
4. Lactobacillus plantarum Lb 329-6 (Valio) 
5. Clostridium tyrobutyricum K8 control. 
Also in these tests the milk samples were inoculated with a spore 
suspension of Clostridium tyrobutyricum K8 isolated from cheese. 
Clostridia were added to the milk to give an initial clostridia content of 
about 1000 CFU/ml. Milk with added clostridium spores but with no added 
Lactobacillus strain was employed as a control. Lactobacilli were added 
from a culture grown in a 1% whey broth for 48 hours. The samples 
containing clostridium and lactobacillus inoculum were run through a 
program simulating a cheesemaking process, whereafter the samples were 
transferred to +200.degree. C. The clostridia contents were determined 
either on Reinforced Clostridial Agar (R.C.M., Lab 23) by the plating 
method or in Tyrobutyricum Broth (Merck 11734) by the MPN (Most Probable 
Number) technique. The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Anticlostridial effect of Lactobacillus strains 
Clostridium tyrobutyricum K8 contents, CFU/ml 
0 h 13 days 26 days 40 days 
52 days 
60 days 
______________________________________ 
LC-705 1000 &lt;10 &lt;10 &lt;10 &lt;10 &lt;10 
LC-10 1000 1600 4000 5600 3.1 .times. 10.sup.4 
1300 
NRRL-B 1000 3.2 .times. 
nt 1.4 .times. 
2.3 .times. 10.sup.4 
1.4 .times. 10.sup.4 
10.sup.4 10.sup.4 
Lb 329-6 
1800 1.2 .times. 
nt 7.9 .times. 
9.5 .times. 10.sup.6 
1.0 .times. 10.sup.7 
10.sup.6 10.sup.6 
K-K8 800 1.4 .times. 
5.6 .times. 
6.8 .times. 
1.7 .times. 10.sup.7 
1.0 .times. 10.sup.6 
10.sup.7 10.sup.7 
10.sup.7 
______________________________________ 
The results show that Lactobacillus rhamnosus LC-705 is the most 
advantageous of the studied strains; it has an excellent inhibitory effect 
on the growth of clostridia. Also the other Lactobacillus rhamnosus strain 
performed better than the studied Lactobacillus plantarum strains, and 
thus Lactobacillus rhamnosus strains are advantageous for use in 
accordance with the invention. 
EXAMPLE 3 
Use of Lactobacillus rhamnosus LC-705 strain as a starter in preparing 
Swiss cheese 
Normal Swiss cheese starters were employed in the cheese preparation. In 
addition, 0.3% of a Lactobacillus rhamnosus LC-705 culture was added to 
the test cheeses. The amount may vary according to cheese variety and 
clostridial spore content. A suitable amount may be for example 1-40 
1/11000 1 of cheese milk or 0.01-0.4%/cheese kettle. Ten clostridium 
spores per litre of milk were added to two test cheeses. The spores were 
Clostridium tyrobutyricum spores, the quantity employed being known to 
produce butyric acid fermentation. 
The LC-705 starter was grown in the cheese dairy as a normal starter; 1% of 
culture was inoculated into the milk and grown at 37.degree. C. for 24 h. 
After culturing, the lactobacillus content was about 3-5.times.10.sup.8 
/ml and the acidity was about 20-25.degree. SH. 
The clostridial, lactic acid, acetic acid, citric acid and butyric acid 
content and pH of the cheeses were determined during the first week at 
one-day intervals and thereafter at intervals of one week. Prepared 
cheeses that had ripened for three months were also evaluated in the 
conventional way by normal quality control, which in addition to chemical 
analyses also included organoleptic evaluation. The results after five 
days and six weeks are shown in Tables 3 and 4. 
TABLE 3 
__________________________________________________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Additions Swiss cheese, 5 days 
batch clostridia/ 
lactic acid 
acetic acid 
citric acid 
No. 
LC-705 
clostr. 
g % mg/100 g 
% pH 
__________________________________________________________________________ 
1. - - &lt;3 1.19 36 0.11 5.52 
2. + - &lt;3 1.27 116 &lt;0.02 
5.46 
3. - + 9 1.21 39 0.10 5.49 
4. + + &lt;3 1.27 122 &lt;0.02 
5.42 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Additions Swiss cheese, 5 days 
batch clostridia/ 
lactic acid 
acetic acid 
butyric acid 
No. 
LC-705 
clostr. 
g % mg/100 g 
% pH 
__________________________________________________________________________ 
1. - - &lt;100 
2. + - &lt;100 
3. - + 2800 0.70 130 15 5.95 
4. + + &lt;100 1.13 150 3 5.62 
__________________________________________________________________________ 
The results show that clostridia can be detected in cheeses with added 
clostridia but with no added LC-705 starter as early as after five days. 
After six weeks, the clostridia content is distinctly higher in cheese 
prepared with added clostridia than in cheese prepared with a 
corresponding addition but also having LC-705 starter added. The amount of 
butyric acid in the cheese with a clostridia addition is above the 
gustative limit. 
The results for ripened cheeses are shown in Table 5. 
TABLE 5 
__________________________________________________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Swiss cheese, 3 months 
Additions entero- 
lactic 
acetic 
propionic 
butyric 
organo- 
batch clostr./ 
cocci/ 
acid 
acid acid acid leptic 
No. 
LC-705 
clostr. 
g g % mg/100 g 
mg/100 g 
mg/100 g 
evalution 
__________________________________________________________________________ 
1. - - &lt;10 5 .times. 10.sup.5 
0.30 
280 400 6 normal 
2. + - &lt;10 8 .times. 10.sup.3 
0.55 
290 300 4 normal 
3. - + 17 000 
4 .times. 10.sup.6 
0.22 
290 400 60 butyric 
acid 
fermen- 
tation 
4. + + &lt;10 8 .times. 10.sup.2 
0.61 
300 310 5 normal 
__________________________________________________________________________ 
The milk used for the tests was of an excellent quality and did not contain 
clostridia to such an extent as to induce natural butyric acid 
fermentation in the cheeses. The cheeses which have added clostridia only 
show butyric acid fermentation. On the other hand, the cheeses which along 
with added clostridia also have added LC 705 starter show no butyric acid 
fermentation. The LC 705 starter thus clearly inhibits clostridial growth 
and prevents butyric acid fermentation. The LC 705 starter also inhibits 
the growth of enterococci in cheese. Enterococci are part of the natural 
bacterial flora that comes into the cheese from milk and from the cheese 
dairy. They are indicative of the level of hygiene, and thus their content 
should be as low as possible. Hence this positive effect is also worth 
paying attention to. 
Also the results of chemical and organoleptic evaluation support the above 
microbiological results. 
EXAMPLE 4 
Use of Lactobacillus rhamnosus LC-705 strain to replace nitrate in cheese 
production 
In this test, an LC 705 starter was used to replace nitrate in the 
production of Edam cheese. Clostridia were added into the cheese milk (20 
spores/1 l of milk) in order to assure butyric acid fermentation. 
The LC 705 starter was grown as described in Example 3 and added into 
kettle milk along with other normal starters in an amount of 0.3-0.35%. 
The amount added may vary according to need. The results for kettle milk 
and cheeses five days old are shown in Tables 6 and 7. The results for 
ripened Edam cheeses six weeks old are shown in Table 8. 
TABLE 6 
______________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Additions Kettle milk 
batch No. 
nitr. LC-705 clostr. clostr./g 
pH 
______________________________________ 
1. + - - &lt;0.3 4.67 
2. + - + 2.3 4.65 
3. - + - &lt;0.3 4.64 
4. - + + 9.3 4.64 
______________________________________ 
TABLE 7 
__________________________________________________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Additions Edam cheese, 5 days 
batch clostr./ 
lactic acid 
butyric acid 
No. nitr. 
LC-705 
clostr. 
g % mg/100 g 
pH 
__________________________________________________________________________ 
1. + - - &lt;3 1.18 0.5 5.40 
2. + - + &lt;3 1.19 0.5 5.39 
3. - + - &lt;3 1.21 0.5 5.39 
4. - + + &lt;3 1.19 0.5 5.40 
__________________________________________________________________________ 
TABLE 8 
__________________________________________________________________________ 
Effect of Lactobacillus rhamnosus on cheese quality 
Additions Edam cheese, ripened 
batch clostr./ 
lactic acid 
butyric acid 
No. nitr. 
LC-705 
clostr. 
g % mg/100 g 
pH 
__________________________________________________________________________ 
1. + - - &lt;3 1.19 4 5.52 
2. + - + &lt;3 1.18 3 5.54 
3. - + - &lt;3 1.25 2 5.45 
4. - + + &lt;3 1.24 3 5.47 
__________________________________________________________________________ 
Thus the effectiveness of the LC705 starter in preventing butyric acid 
fermentation in all test cheeses is equal to or even better than that of 
nitrate.