METHOD FOR INHIBITING FUNGI

Abstract: A method for providing fungal inhibition using Lactobacillus casei var. rhamnosus which produces an antifungal substance is described. Bacterial compositions of LCR are also described. The method and compositions are particularly useful for producing fermented foods, and for treatment of silage.

Description: BACKGROUND OF THE INVENTION (1) Field of the Invention The present invention relates to a method and composition using or containing a Lactobacillus having the identification characteristics of Lactobacillus casei var. rhamnosus NRRL-B-15972 and having antifungal properties. In particular the present invention relates to the use of the Lactobacillus in foods. (2) Prior art The use of lactic acid producing bacteria in methods for producing fermented foods is well known to those skilled in the art. Thus for instance fermented meats (e.g. sausages) and vegetables (e.g. pickles) are all produced using these methods. Members of the genus Lactobacillus have been used extensively for this purpose. It is believed strains of Lactobacillus which produce fungal inhibiting substances are unknown to those skilled in the art. Thus a strain which produces an antifungal substance would be very useful in foods and also in or on other materials, such as living plants, where fungi are a problem. The Food Science Technology Abstracts 82-12-b0211, Vol. 57, No. 1, pp. 8-18 (1982) discloses that Lactobacillus casei var. rhamnosus can be used for preventing spoilage of certain foods. However, it is not known how the Lactobacillus exerts its spoilage preventing activity. Objects It is therefore an object of the present invention to provide compositions including a Lactobacillus which inhibits fungal growth. Further it is an object of the present invention to provide a method which uses this Lactobacillus for the inhibition of fungal growth. These and other objects will become increasingly apparent by reference to the following description. General Description The present invention relates to a method of treating a material which comprises incorporating in or on the material living cells of Lactobacilus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. Further the present invention relates to a storage stable biologically pure culture of Lactobacillus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. Essentially any material can be treated with the antifungal Lactobacillus casei var. rhamnosus NRRL-B-15972. It is preferred that the material be a food and that the food be fermented, since the antifungal Lactobacillus casei var. rhamnosus NRRL-B-15972 of the present invention generates lactic acid in situ from glucose and other sugars present or introduced into the foods. The present strain is particularly useful where the fermented food is prone to microbial spoilage such as is encountered with silage. Plants can be treated with this Lactobacillus to prevent fungal infection. Other materials such as plastics or rubber can be protected by combining the cells with these materials. The material or food should contain at least about 10⁵ cells per gram. Preferably the range is between about 10⁶ and 10⁹ cells per gram. The antifungal substance is cell associated, so a sufficient population is needed to prevent fungal growth. The antifungal Lactobacillus casei var. rhamnosus can be stored in frozen or lyophilized form as is well known to those skilled in the art. The frozen form is preferred using freezing stabilizing agents such as glycerol or xanthan gum because lyophilization causes some die-off of the bacteria. The concentrates normally contain between about 10¹⁰ and 10¹² cells per gram. Plasmids and genetic material in the antifungal Lactobacillus casei var. rhamnosus NRRL-B-15972 can be transferred to other species of Lactobacillus. Any Lactobacillus having the essential antifungal characteristics of the strain NRRL-B-15972 is included within the scope of the present invention. Specific Description Lactobacillus casei var. rhamnosus NRRL-B-15972 has the following characteristics set forth in Table I: EXAMPLE I It was found that Lactobacillus casei var. rhamnosus NRRL-B-15972 could be frozen without problem and with virtually no die-off of cells. The frozen cells had a long shelf life (6 months) with little die-off. The lyophilization characteristics of Lactobacillus casei var. rhamnosus NRRL-B-15972 with different additives was also studied. (1) Lactobacillus casei var. rhamnosus NRRL-B-15972 was grown overnight at 35°C in MRS (Difco, Detroit, Michigan) broth (1000 mL), centrifuged and resuspended in 90 mL of spent medium (10 times culture). (2) Eleven mL of 10X culture was introduced into lyophilization bottle and the pH adjusted to 5.8 with NaOH. (3) The following additives each were added to the bottles: The 10X culture was mixed with each additive and lyophilized 6 hours after freezing in a dry ice and methanol bath. Viable counts were taken on the cells before and after concentration and after lyophilization. The results were as follows: Overnight culture = 3.1 x 10⁹ CFU (Colony Forming Units) 10X culture = 2.5 x 10¹⁰ CFU Lyophilized Cultures Control = 1.37 x 10¹⁰% die off = 43.2 2% MSG = 1.54 x 10¹⁰% die off = 38.4 1% MSG = 1.41 x 10¹⁰% die off = 43.6 2% NFDM = 1.31 x 10¹⁰% die off = 47.6 1% NFDM = 1.93 x 10¹⁰% die off = 30.8 2% MD = 1.34 x 10¹⁰% die off = 46.4 1% MD = 1.19 x 10¹⁰% die off = 52.4 The best results here were obtained by using 2% MSG and 1% NFDM, respectively. Maltodextrin was no better than the control. Either 2% MSG or NFDM is suitable for lyophilization; however, other well known lyophilization agents can be used. EXAMPLE II The effect Lactobacillus casei var. rhamnosus NRRL-B-15972 has on mold growth when grown on cucumber agar was determined. The procedure was as follows: (1) Sterile tempered 10% (w/v) Noble agar was added to sterile filtered cuke juice 5% NaCl (w/v) to produce about 80% (v/v) cuke agar. (2) The plate was spread with approximately 100 colonies of LCR on plates and incubated at 35°C for 72 hours. The colonies were not visible until after 36 hours and had a good size at 72 hours. (3) The cuke agar was overlayed with 0.75% (w/v) cuke agar with the following mold: (1) Penicillium oxalicum , (2) Chaetomium olivacium , (3) Aspergillus terreus , (4) Verticillium sp . It was found that after 72 hours there was no inhibition of P . oxalicum . There was retardation of growth of Aspergillus terreus and Verticillium sp and where Lactobacillus casei var. rhamnosus NRRL-B-15972 colonies were concentrated in one region of plate, there was a total inhibition. There was total inhibition of Chaetomium olivacium . After 5 days, the results were the same. Conodial development was stopped in Aspergillus terreus . This experiment showed that Lactobacillus casei var. rhamnosus NRRL-B-15972 does inhibit some molds in a cucumber medium. It did not inhibit P . oxalicum and only partially inhibited Aspergillus and Verticillium . Chaetomium olivacium was completely inhibited. EXAMPLE III Lactobacillus casei var. rhamnosus NRRL-B-15972 is a rapid grower and as a mold and yeast inhibitor is a useful organism for the pickle industry. Mold and yeast growth on the surface of fermenting cukes are particularly bothersome. This experiment evaluated the growth of LCR in cucumber juices at two temperatures. Its mold inhibiting properties were also evaluated. It was compared with a commercial lactic acid producing culture, Pediococcus pentosaceus , used for this purpose. The procedure was to puree 10 cucumbers in a blender. The puree was centrifuged and poured through cheese cloth under a vacuum. Chlorophyl was removed by centrifugation. NaCl was added to 5% concentration. The yield was 1660 mL of cucumber juice. The juice was filter sterilized through a 0.45 micron filter for immediate use. The remaining juice was frozen until ready for use. The juice had a pH of 5.60 and contained 0.28% L-malic acid. LCR was added to 10 ml of pure juice and 60% juice to give 1 x 10⁶ CPU/mL. The same procedure was used with Pediococcus pentosaceus NRRL-B-11,465. The unprocessed juice was incubated at 25°C and at 18°C. The cuke juice contained 6mL cuke juice; 0.5 mL of a 1:100 dil. Lactobacillus casei var. rhamnosus NRRL-B-15972 or Pediococcus pentosaceus NRRL-B-11,465; 0.34 ml 5M NaCl; and 3.16 mL dH₂O (distilled water). Table II shows Lactobacillus casei var. rhamnosus NRRL-B-15972 is a very rapid fermenter and achieved lower pH's than Pediococcus pentosaceus NRRL-B-11,465. LCR LCR Pediococcus pentosaceus Pediococcus pentosaceus NRRL-B-11,465 (100% juice) (60% juice) (100% juice) (60% juice) growth at 25°C ++ ++ + + growth at 18°C + + ± ± pH 65 hours 25°C 3.86 3.78 4.37 4.32 pH 65 hours 18°C 4.97 4.40 5.65 5.54 pH 90 hours 25°C 3.75 3.71 4.15 3.95 pH 90 hours 18°C 4.23 4.01 5.27 4.64 The + or - designations have the following meanings: ++ fast growth + moderate growth ± marginal growth In order to evaluate mold inhibition after 90 hours growth, 1 drop of a P . oxalicum spore suspension was added to each tube. A control tube of 100% cuke juice was adjusted to pH 3.77 with 85% lactic acid before the spores were added. The tubes were at 10°C for 14 days. The results are shown in Table III Mold growth 60% PP 25°C ++++ 8°C ++ Control +++ 60% LCR 25°C + 18°C +± 100% PP 25°C + 18°C ++++ 100% LCR 25°C ± 18°C +± + = some growth ++++ = rapid growth Lactobacillus casei var. rhamnosus MRRL-B-15972 grew more rapidly and lowered the pH faster than Pediococcus pentosaceus NRRL-B-11,465 in 100% and 60% juice at 25° and 18°C. Also Lactobacillus casei var. rhamnosus NRRL-B-15972 appeared to be more effective in inhibiting this mold even though it was not effective in Example II against the same fungi.

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A method of treating a material to inhibit fungal growth which comprises incorporating in or on the material living cells of Lactobacillus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. The method of Claim 1 wherein the material is a food and wherein the cells are maintained as living cells in the food. The method Claim 2 wherein the food is fermented by the Lactobacillus . The method of Claim 2 wherein at least about 10⁵ cells per ml are introduced into the food. The method of Claim 2 wherein between about 10⁶ and 10⁸ cells per gram of material are incorporated. A storage stable biologically pure culture of Lactobacillus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. The culture of Claim 6 containing at least about 10¹⁰ cells per ml of the Lactobacillus . The culture of Claim 6 in a frozen form so that the culture is storage stable. The culture of Claim 6 in a lyophilized form so that the culture is storage stable. The culture of Claim 6 containing at least about 10⁶ to 10¹² cells per ml. Claims for the following Contracting State : AT A method of treating a material to inhibit fungal growth which comprises incorporating in or on the material living cells of Lactobacillus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. The method of Claim 1 wherein the material is a food and wherein the cells are maintained as living cells in the food. The method Claim 2 wherein the food is fermented by the Lactobacillus . The method of Claim 2 wherein at least about 10⁵ cells per ml are introduced into the food. The method of Claim 2 wherein between about 10⁶ and 10⁸ cells per gram of material are incorporated. A storage stable biologically pure culture of Lactobacillus casei var. rhamnosus NRRL-B-15972 which inhibits the fungal growth. The culture of Claim 6 containing at least about 10¹⁰ cells per ml of the Lactobacillus . The culture of Claim 6 in a frozen form so that the culture is storage stable. The culture of Claim 6 in a lyophilized form so that the culture is storage stable. The culture of Claim 6 containing at least about 10⁶ to 10¹² cells per ml. A process for preparing a storage stable biologically pure culture of Lactobacillus casei var. rhamnosus NRRL-B-15972 which produces an antifungal substance and inhibits the fungal growth by freezing or lyophilizing the antifungal Lactobacillus casei var. rhamnosus . The process of claim 11 wherein the culture contains at least about 10¹⁰ cell per ml of the Lactobacillus . The process of Claim 11 wherein the Lactobacillus is frozen. The process of Claim 11 wherein the Lactobacillus is lyophilized. The process of Claim 6 wherein the culture contains at least about 10⁶ to 10¹² cells per ml.

Inventors: FOWLER, GEOFF G., KING, STEPHEN W., VANDENBERGH, PETER A.
Applicants: MICROLIFE TECHNICS, INC.
Application year: 1986
Main classification: A23L3
IPC codes: C12N1, A23L3, A23C19, A23K3