Inhibition of clostridia with lactic acid bacteria

Abstract: 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.

Description: 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, spore-forming bacteria with a fermentative metabolism. Clostridia are very effective producers of gas (H 2 and CO 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 2 and CO 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 acid-forming 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. Patent 4 584 199. U.S. Patent 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 delbrückii 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 delbrückii 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. "Alimenta" 5 (1971) 167-180 discusses the use of a number of different Lactobacillus variants in the inhibition of Clostridia tyrobutyricum , for the purpose of preventing late blowing in hard cheese. 180 different L . casei strains were included in tests, including some of the variant L. casei rhamnosus . However, the vast majority were inactive, and only 3 strains were found to be inhibitory. This compares with 7 strains which were found to promote Cl . tyrobutryricum activity, and 170 which were inert. Individual L. rhamnosus strains are not identified. EP-A-0,302,300 relates to methods for retarding yeast and mould growth in the food industry, for example to extend the shelf life of cottage cheese. The method involves the use of Lactobacillus casei var. rhamnosus NRRL-B-15972 to inhibit Penicillium oxalicum spores. J. Food Science 55 (1990) 386-390 discloses the use of various Lactobacilli for the control of acidity during cheese making. The only example of L. rhamnosus tested was strain L2F, which was found to lead to the undesirable effects of high acidity, bitterness, off-flavour, openness and crumbling. There is no mention of Clostridia inhibition. The applicants own earlier application EP-A-0,576,780 relates to the novel strain Lactobacillus casei ssp rhamnosus LC-705, DSM 7061, but this strain is only disclosed as being effective against moulds and yeasts, such as Penicillium , Aspergillus , Cladosporium , Fusarium and Candida . There is no suggestion of utility against any bacteria. 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 LC-705, DSM 7061 has an excellent anticlostridial effect. Thus the present invention relates to the use of Lactobacillus rhamnosus LC-705, DSM 7061 in the food industry for inhibition of clostridia. Preferably, Lactobacillus rhamnosus LC-705, DSM 7061 is added to food. The present invention further relates to a method for preventing butyric acid fermentation, the method being characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used to inhibit the activity of clostridia. The invention also extends to a method for preparing cheese, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used in the preparation of the cheese. Detailed Disclosure of the Invention The present invention, therefore, relates to the use of Lactobacillus rhamnosus LC-705, DSM 7061 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 diary industry, canning industry, the meat industry, and the 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 the organism Lactobacillus rhamnosus LC-705, available at the microorganism collection of Valio Oy by product number LC-705. 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°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%. In accordance with the invention, 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 LC-705, DSM 7061 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°C with or without pH adjustment. For example, the preparation of Lactobacillus rhamnosus LC-705 has been described in detail in the above mentioned EP-A-0,576,780. The cell-containing fermentation broth is recovered and can be used as such, concentrated, or lyophilized. The lyophilization is preformed 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 LC-705 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. Simultaneously 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 preparations, 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 LC-705 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. The anticlostridial Lactobacillus rhamnosus strain 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. 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 Lactobacillus rhamnosus LC-705 strain The test series was conducted with milk prepared from 10% milk powder (à 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 cheese-making process, whereafter the samples were transferred to +20° 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. 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 1. LC-705 1% 9.8x10 3 4.9x10 3 <10 <10 <10 <10 2. Control K-K8 8.6x10 3 1.1x10 3 1.8x10 4 2.4x10 5 2.3x10 4 8.1x10 4 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 +20° 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. Anticlostridial effect of different Lactobacillus strains Clostridium tyrobutyricum K8 contents, CFU/ml 0 h 13 days 26 days 40 days 52 days 60 days LC-705 1000 <10 <10 <10 <10 <10 LC-10 1000 1600 4000 5600 3.1x10 4 1300 NRRL-B 1000 3.2x10 4 nt 1.4x10 4 2.3x10 4 1.4x10 4 Lb 329-6 1800 1.2x10 6 nt 7.9x10 6 9.5x10 6 1.0x10 7 K-K8 800 1.4x10 7 5.6x10 7 6.8x10 7 1.7x10 7 1.0x10 6 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. 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 l 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°C for 24 h. After culturing, the lactobacillus content was about 3-5 x 10 8 /ml and the acidity was about 20-25 °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 were shown in Tables 3 and 4. Effect of Lactobacillus rhamnosus on cheese quality Additions Swiss cheese, 5 days batch No. LC-705 clostr. clostridia /g lactic acid % acetic acid mg/100 g citric acid % pH 1. - - <3 1.19 36 0.11 5.52 2. + - <3 1.27 116 <0.02 5.46 3. - + 9 1.21 39 0.10 5.49 4. + + <3 1.27 122 <0.02 5.42 Effect of Lactobacillus rhamnosus on cheese quality Additions Swiss cheese, 6 weeks batch No. LC-705 clostr. clostridia /g lactic acid % acetic acid mg/100 g butyric acid mg/100 g pH 1. - - <100 2. + - <100 3. - + 2800 0.70 130 15 5.95 4. + + <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. Effect of Lactobacillus rhamnosus on cheese quality Additions Swiss cheese, 3 months batch No. LC-705 clostr. clostr. /g enterococci /g lactic acid % acetic acid mg/100g propionic acid mg/100g butyric acid mg/100g organoleptic evaluation 1. - - <10 5x10 5 0.30 280 400 6 normal 2. + - <10 8x10 3 0.55 290 300 4 normal 3. - + 17 000 4x10 6 0.22 290 400 60 butyric acid fermentation 4. + + <10 8x10 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. Effect of Lactobacillus rhamnosus on cheese quality Additions Kettle milk batch No. nitr. LC-705 clostr. clostr. /g pH 1. + - - <0.3 4.67 2. + - + 2.3 4.65 3. - + - <0.3 4.64 4. - + + 9.3 4.64 Effect of Lactobacillus rhamnosus on cheese quality Additions Edam cheese, 5 days batch No. nitr. LC-705 clostr. clostr. /g lactic acid % butyric acid mg/100g pH 1. + - - <3 1.18 0.5 5.40 2. + - + <3 1.19 0.5 5.39 3. - + - <3 1.21 0.5 5.39 4. - + + <3 1.19 0.5 5.40 Effect of Lactobacillus rhamnosus on cheese quality Additions Edam cheese, ripened batch No. nitr. LC-705 clostr. clostr. /g lactic acid % butyric acid mg/100g pH 1. + - - <3 1.19 4 5.52 2. + - + <3 1.18 3 5.54 3. - + - <3 1.25 2 5.45 4. - + + <3 1.24 3 5.47 Thus the effectiveness of the LC-705 starter in preventing butyric acid fermentation in all test cheeses is equal to or even better than that of nitrate.

Claims: Use of Lactobacillus rhamnosus LC-705, DSM 7061 in the food industry for inhibition of clostridia. Use as claimed in claim 1, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used in cheese production. Use as claimed in claim 1 or claim 2, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used to prevent butyric acid fermentation. Use as claimed in any one of claims 1-3, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used as a starter. Use as claimed in any preceding claim, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is added to food. A method for preventing butyric acid fermentation, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used to inhibit the activity of clostridia. A method for preparing cheese, characterized in that Lactobacillus rhamnosus LC-705, DSM 7061 is used in the preparation of the cheese.

Inventors: MAEYRAE-MAEKINEN ANNIKA, SUOMALAINEN TARJA, MAEYRAE-MAEKINEN, ANNIKA, SUOMALAINEN, TARJA, Mäyrä-Mäkinen, Annika 
Applicants: VALIO OY
Application year: 1995
Main classification: A23L3
IPC codes: A23C19, A23L3