CA1269942A - Method and inoculant for preserving agricultural products for animal feed - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a method of preserving agricultural products comprising treating these pro-ducts with an effective amount of Lactobacillus plantarum ATCC 53187 or mutants thereof and the treat-ing organism.

Description

~Z69~342 TITLE OF THE INVENTION:

METBOD AND INOCUL~NT FOR
PRESERVING AGRICULT~RAL PRODUCTS
FOR ANIMAL FFED

BPCKG~O~D OF THE INVE~TION

Field of the Invention The invention relates to a method for preserving agricultural products which are used for animal feed and a Lactobacillus plantarum inoculant for use in the method.

Brief ~escription of the Backaround ~rt Spoilage of stored agricultural products is a major problem in the farming industry. This spoilage often arises during long-term storage of these aqri-cultural materials. Typically, long-term storage is necessary to provide adequate supplies of feed for domestic animals during those times of the year when fresh feeds are unavailable.
The storage of silage, baled hay, and storaae of high moisture grain are among the most common approaches for providing an adequate supply of feed for those months when fresh feeds are unavailable.
~ ilage is produced by the anaerobic fermentation of the materials described above. Typically, the material i~ veaetable matter which is cut and then ~`

~ -~2 9942 loaded into a silo, most commonly a vertical cylin-drical structure, and kept anaerobic to inhibit the growth of aerobic spoilage organisms. Ideally, under anaerobic conditions the homofermentative bacteria proliferate, producing lactic acid which, by lowering the pH of the environment, further inhibits the qrowth of undesirable organisms. Unfortunately, the contents of the silo often become exposed to air due to leaks in the silo structure or during the gradual removal of silage. Such re-exposure to aerobic conditions allows proliferation of fungi to occur and results in spoil-age of the silage.
In most circumstances, hay is cut and allowed to dry in the field until the moisture level has dropped to 8-15%. This drying process is performed because hay baled at higher levels of moisture is much more likely to degenerate due to th`e influence of heat of decomposition and the activity of epiphytic spoilage organisms. Unfortunately, while drying the hay to 8-15% moisture substantially avoids these detrimental effects, these low levels of moisture resul, in high field losses due to drying and hay of diminished nutritional value due to loss of the leafy ~art of the plant. It is, therefore, most desirable to bail hay at moisture levels of 25-30% if deterioration due to heat and microorganisms can be controlled.
The high moisture grain is stored in silos under anaerobic conditions, as described above for silage.
Spoilage of high-moisture corn is due primarily to funai which proliferate under storaae conditions.
~andida auilliermondii, Candida ~elliculosa, ~ansemula __ _ _ _ _ _ _ _ _ anomala, Penicillium s~p., and ~speroillus sp~. are .

among those fungi often responsible for the spoilage of high-moisture orains and silaae.
Often the materials which are preserved using these processes contain naturally-occurring o~portu-nistic epiphytes. These epiphytes may be beneficial or detrimental in determining the eventual status of the preserved agricultural material and compete among themselves for ecological dominance in the stored material.
The predominance of undesirable organisms can result in spoilage and contamination of the feed material such that it can no lonaer be used to main-tain domestic animals. These undesirable organisms may exert their effect by destroying the nutritive value of the feed, or even by producing toxins which are dangerous to the well-being of the animals. A
common source of s~oilage organisms is found in stor-age facilities and on associated processino equipment.
These spoilage organisms may be either bacteria or fungi. Common bacterial spoilaoe organisms are those which are members of the genera Clostridium and Listeria. Fungal spoilage is often caused by members of the genera ~sperqillus, Candida, Mucor, and Sac-charomyces.
Various approaches have been made to limit the proliferation of those epiphytes and storage organisms responsible for spoilage of agricultural products.
Since the de~irable epiphytes are tolerant of low pH, while spoilage organisms such as Clostridium ~ are not, one simple approach has been to spray the agri-cultural material at the time of storage with an acid as, for example, propionic acid. ~ major disadvantage ~2605 073185 lZ69942 of this approach is its cost since large quantities of acid must be used to adequately treat the agricultural material. In addition, the inherently toxic and cor-rosive nature of propionic acid requires that special handling techniques be used by the farmer to protect personnel handling the acid. ~urther, all equipment coming in contact with the acid must be thoroughly decontaminated to avoid corrosive damaae.
Another approach to limiting the arowth of unde-sirable organisms during the storaae of aaricultural materials has been to inoculate these materials with those epiphytes thought to be beneficial to the pre-servation of the agricultural material such that the epiphytes will be able to out-compete and thereby, limit the proliferation of the spoilage organisms.
Those organisms which were considered most beneficial in the past nave been selected primarily on the basis that they were homofermentative lactic acid producers.
The rationale for this approach is that the low pH
caused by the lactic acid produced by these organisms would inhibit the growth of spoilage organisms.
Strains from species of the genera Lactobacillus, Streptococcus, and Pedicoccus are among those consid-ered to be most beneficial in achieving this tyDe of inhibitory effect.
Lactic acid bacteria have often been used in the preservation of food products utilizing fermentation processes. For example, Jeffreys, United States Patent 3,677,897, discloses a method of culturing and preserving lactic acid bacteria for use as starter cultures in various fermentation Drocesses. The patentee states that the method of the invention can ~Z699g2 be used to stabilize any of the lactic acid bacteria, singly or in combination, which are well known in the dairy and fermentation industries. ~mong those organ-isms which are mentioned is actobacillus E~antarum.
The method comprises coating the organisms with an acetylated monoglyceride and adding a carrier selected from the group consisting of modified cellulose and modified starches as, for example, bran solids.
In Moon (Journal of Science, Food & Aqriculture, 32: 675 (1981)), the author studied the effect of experimental silage prepared from green bean and potato processing wastes which were inoculated with a strain of Lactobacillus plantarum. Since these materials were very high in moisture, it was necessary to add ground peanut hulls to reduce the percentage of moisture prior to inoculationO~ In producing silage in this manner, the author reports that large numbers of undesirable organisms were only present in the uninoc-ulated control silages and that overall the addition of ~. plantarum had a beneficial effect on the fermen-tation of this silage material.
Woolford et al. (Grass & Forage Scie ce, 39: 139 k (1984)) describes studies on the effect of different cultures of lactic acid bacteria on silage fermenta-tion. The authors set up experimental silages and evaluated 21 strains of lactic acid bacteria from various genera and species, in terms of meeting cer-tain criteria which the authors believe are indicative of the ability to produce good silage. The authors state that none of these cultures satisfied all of the criteria, but three strains (Stre~tococcus durans, strain 1024; Lactobacillus acido~hilus, strain 2356;

~2605 073185 `- 1Z699~

and Lactobacillus plantarum, strain 6) had greater potential for producing good farm silage than the other 18 strains tested.
In a study reported by McMahon et al. (~pplied Microbioloqy, 30: 103 tl975)), the authors studied the deterioration of high-moisture corn in leaky silos which were filled with either normal high-moisture corn ~HMC) or with HMC severely infested with Southern corn leaf blight fungus (Helminthosporium _aydis). The authors determined the number of mesophilic bacteria, lactobacilli, coliforms, yeast and molds present on the corn samples as received and periodically during 220 days of storage. The authors state that Lacto-bacillus plantarum was the most frequent bacterial isolate, although it was not as predominant in the normal corn as had been found in a previous study.
Indeed, L. plantarum was found at greater frequency in the blight infested corn than in the normal corn.
In Dellagio et al. (S~YStemic ~ Ppplied Microbiol_-~, 5: 534 (1984)), the authors studied the lactic acid bacteria present in high-moisture corn grain for up to 120 days using DN~-DN~ hybridization. The authors determined that all of the homofermentative lactobacilli tested were highly related to the type strain of Lactobacillus vlantarum.
The American Type Culture Collection has publicly available a strain of Lactobacillus E~ntarum (ATCC
14431) which was isolated from grass silage.
However, in spite of extensive research into the isolation and development of various organisms for use as agricultural product inoculants, spoilage of these products has remained an on-going problem.

~Z~9g42 SUMMARY OF THE INVENTION
In the present invention, agricultural products that are to be preserved as a feed are treated with an effective amount of Lactobacillus plantarum ATCC
53187. ~nlike most members of this species, L. ~lan-tarum ATCC 53187 is capable of inhibiting common fungal spoilage organisms that are known to be respon-sible for the deterioration of agricultural products stored for long periods of time for use as feeds. It is possible that the inhibition of these fungi and molds is caused by the production of a toxin or anti-biotic-like substance which causes a fungistatic or fungicidal effect on these organi~ms.
Thus, the present invention provides a method of treating agricultural products to enhance their pres-ervation which comprises administering to the agricul-tural products an effective amount of Lactobacillus plantarum ATCC 53187 or mutants thereof.
The invention also provides the T,actobacillus strain and effective mutants thereof.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS
Spoilage of agricultural products due to deteri-oration caused by the growth of harmful fungi is a major problem in the agricultural community. In the method according to the invention, agricultural pro-ducts are treated with an effective amount of Tacto-bacillus plantarum ATCC 53187, or mutants thereof, to enhance preservation of the ~roducts and diminish the proliferation of spoilaae organisms.
The method of the invention is especially useful in that the oraanism of the invention is non-~atho-genic and thereby will not cause disease in an animal consuming the preserved agricultural product. Unlike many types of preservation which util;ze the applica-tion of expensive or hazardous chemicals, the method of the invention relies instead uDon the enhancement of coloni~ation by desirable organisms through improv-ing their ability to compete with naturally-occurring spoilage organisms. Controlling these harmful organ-isms is a major consideration, since many of the organisms associated with spoilage of preserved agri-cultural products are fungi which are known to produce potent mycotoxins. These mycotoxins are known to cause illness and even death in animals consuming feed contaminated with these fungi.
The organism which is effective in enhancing the preservation of agricultural products is Lactobacillus plantarum ATCC 53187 or effective mutants thereof.
I,actobacillus ~ntarum ~TCC 53187 is on deposit at the American Type Culture Collection, Rockville, Maryland and has the ATCC ~ccession ~o. 53187. By the term "effective mutants thereof" is intended any and all mutants of ~. plantarum ATCC 53187 which demon-strate the desired fungal inhibiting properties of the present strain or substantial equivalents thereof.
Such mutants are considered to be functionally equiva-lent to the parental strain. L. plantarum ATCC 53187 was isolated from corn silage using techniques well known in the art.
It is well known to those of ordinary skill in the art that spontaneous mutation is a common occurrence in microorganisms and that mutations can also be in-tentionally produced by a variety of known techniques.
For example, mutants can be induced using chemical, radioactive, and recombinant techniques. Chemical ~2605 073185 g mutagens can be divided into four main qroups based upon their mode of activity. The four groups and examples of each are set out in Table 1 below.

ACTIVITY EXAMPBES
Base analogues 5-bromouracil, 2-amino-purine Deaminating agents nitrous acid, hydroxyla-mine Alkylating agents ethyl ethanesulfonate, nitrosoguanidine Acridine derivatives Acridine orange, ethidium bromide Radiation-induced mutations can be caused by such agents as ultraviolet light and x-rays. The primary mechanism by which mutations may be caused result from excision or post-replication repair by recombination.
In addition, mutations can also be produced by recom-binant techniques using restriction endonucleases. The utilization of this technique is especially valuable in allowing the deletion or insertion of large DNA
fragments. The present invention includes within its scope mutants of L. ~lantarum ~TCC 53187 produced utilizing any of the mutagens known to the art.
Regardless of the manner in which the mutations are induced, the critical issue is that the mutants function to preserve the agricultural product as described for the parent strain. In other words, the present invention includes mutations resulting in such ~269942 minor changes as, for example, minor taxonomic altera-tions such as the fermentation of certain sugars.
The agricultural products which are effectively preserved by the present invention include all agri-cultural products in which deterioration due to the activity of spoilage organisms is a potential problem.
Among the agricultural products which can be treated using the present invention are silage, baled hay and high-moisture grain. By the term ~silage" is intended animal feeds prepared from agricultural products that can be derived from animal or vegetable matter or mixtures thereof. The animal matter which is utilized can be derived from fish, poultry, or cattle. Most commonly, silage is derived from the fermentation of vegetable material such as, for exam-ple, grasses, legumes, whole-crop cereals, potatoes, or food manufacture wastes. Commonly ensiled grasses are rye grass, timothy, cocksfoot, and fesque. Exam-ples of legumes which can be used to produce silage are alfalfa, peas, clover, lucerne, sainfoin, field bean, common vetch, and lupin. Whole-crop cereals which can be ensiled include corn, sorahum, barley, wheat, oats, and rye. It is also possible to produce silage using waste material derived from food manu-facturing processes such as, for example, those derived from sugar cane, sugar beets, peas, or fruit.
By the term "hay" is intended all forms of hay as the term is commonly used in agriculture. Hay is most commonly composed of alfalfa, or mixtures of alfalfa and grass.
By the term "high-moisture grain" is intended corn in the form of whole shelled corn, or ground or rolled shelled corn.

~.269~4;2 In the present invention, the inhibition of fungi responsible for spoilage of agricultural products such as silage, hay, and high-moisture grain, is accom-plished by treating the agricultural material with L.
E~l___rum ATCC 53187 or compositions containing this organism, as well as treatment with effective mutants of L. plantarum ATCC 53187 and compositions containing same.
The compositions which are used in the method of the invention may be in either liquid or dry form and may contain additional bacterial strains. In solid treatment forms, the composition may comprise the organism of the invention together with a carrier. The carrier may be in the nature of an aqueous or non-aqueous liquid or a solid. In solid forms, the compo-sition may contain such diluents as calcium carbonate, starch, or cellu]ose. The solid composition can be applied directly to the agricultural product or can be first dispersed in a liquid suspension and sprayed thereon.
Typical compositions useful for treating products according to this invention contain 102-1012 viable organisms/gm, preferably 104-101 viable organisms/gm, and more preferably 106-103 viable organisms/gm.
The treatment range for an agricultural product is typically 105-1015 viable organisms/ton, preferably 107-1013 viable organisms/ton, and more preferably 109-1011 viable organisms/ton.
The compositions of the present invention can include, in addition to Lactobacillus plantarum ATCC
53187 or mutants of this organism, such other common farm product preservation organisms as, for example, Streptococcus spp. or Pediococcus spp.

Those or ordinary skill in the art will know of other suitable carriers and dosaqe forms, or will be able to ascertain such, using routine experimentation.
Further, the administration of the various composi-tions can be carried out using standard techniques common to those of ordinary skill in the art.
The above disclosure ~enerally describes the present invention. A more complete understanding can be obtained by reference to the following specific exa~ples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

Lactobacillus plantarum (ATCC 53187) is a gram-positive rod which was isolated from corn silage. The fermentation profile of this organisms is shown in Table 2. This profile was performed using a com-mercially available test system ~CHL kit, ~MS Labora-tories, Darts Mill, Flemington, New Jersey1.

* Trad~k .. ,., ,, :
~ A2605 073185 *.. ~,~. , .. I

12699~2 Table 2 SUBSTRATE RE~CTION SUBSTRATE RF~CTION
Glycerol - Salicin +
Erythritol - Cellobiose +
D-Arabinose - Maltose +
L-Arabinose + Lactose +
Ribose + Melibiose +
~-Xylose - & ccharose +
LrXylose - Trehalose +
Adonitol - Inuline E-Methyl-xyloside - Melezitose +
Galactose + D-Raffinose +
O-Glucose + Starch D-Fructose + Glycogen D-Mannose + Xylitol L-Sorbose - beta-Gentiobiose +
Rhamnose - D-Tùranose Dulcitol - D-~yxose Inositol - D-Tagatose Mannitol + D-Fucose Sorbitol + L-Fucose alpha-Methyl D-nannoside - D-Arabitol alpha-Methyl D-glucoside - L-Arabitol N-~cetyl-glucosamine + Gluconate +
Amygdalin + 2-Keto-gluconate Arbutin + 5-Keto-glucoate Esculin +

9~4;~

Strain ATCC ~3187 was grown in MRS broth (DIFCO) supplemented with DL-threonine (Chassy et al., Journal of Bacterioloqy, 127: 1576, 1976) and the plasmid profile determined as described by Anderson et al., Applied & Environmental Microbioloqy, 46: 549 (1983).
The size of the plasmids was established using hori-zontal electrophoresis in agarose tO.6%~ with a TRIS-borate buffer (Myers et al., Journal of sacteri-oloqy, 127: 1529 (1976)). ATCC 53187 was found to have plasmids of 3.5 and 35.0 me~adaltons.

EXAMPL~ 2 The ability of L. plantarum ATCC 53187 to inhibit fungi known to cause spoilaae in aaricultural products was compared to that of 18 other strains of ~. Plan-tarum. These strains were i,solated from vegetable material that can be used to produce preserved agri-cultural feedstuffs. The indicator mold used in the experiment was isolated from spoiled high-moisture corn and was identified as being from the genus Mucor.
All isolation techniques used are common to those of skill in the art.
In order to measure the anti-mycotic activity of a given strain of L. plantarum, the organism was asep-tically streaked down the center of an ~RS agar plate (MRS broth (DIFCO) with 0.15% Bacto agar (DIFCO)), after anaerobic incubation at 37C for 48 hours to allow growth of the Lactobacillus. The funqal indi-cator organism was inoculated into 5 ml of sterile, melted (48C) Potato ~extrose ~aar (DIFCO) which was poured onto the surface of the MRS aaar plate. The overlay agar containing the fungal indicator organism was allowed to solidify at room temperature for 5-10 * Trademark lZ69942 minutes. The plates were then incubated aerobically at 37C and examined for zones of inhibition on days 4 and 7. The results of this testing are shown in Table 3.

~2605 073185 ~Z69942 Table 3 Mold Inhibition By Various Strains of ~. plantarum Strain Source Inhib_tiona_ Day 4 Day 7 ATCC 53187 corn 27 22 S80 feces 0 0 C54 corn 0 0 alpha 5 alfalfa 0 0 C55 corn 24 21 345 alfalfa 0 346 alfalfa 8 0 347 alfalfa 0 0 287 corn 0 0 SC13A corn 5 0 SC18A corn 0 0 639A grass 10 0 640A grass 3 0 15B alfalfa 10 0 57B unknown 3 0 SC30B corn 0 0 84B corn 0 0 121B corn 0 0 97A artichoke 0 0 a mm ~lthough several strains of ~. plantarum displayed early transient inhibition on Day 4, only one strain (C55) other than strain ATCC 53187 displayed persis-tent anti-mycotic activity as indicated by the zones of fungal growth inhibition on Day 7. The results of -- ~;26g~4 ~

this testing show that the ability of a given strain of L. plantarum to show anti-mycotic activity cannot be predicted.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

~2605 073185

Claims (17)

WHAT IS CLAIMED AS NEW AND IS DESIRED TO BE COVERED BY
LETTERS PATENT IS:

1. A method of preserving an agricultural pro-duct which comprises treating said product with an effective amount of Lactobacillus plantarum ATCC 53187 or effective mutants thereof.

2. The method of claim 1, wherein said agricul-tural product is selected from silage, baled hay, and high-moisture grain.

3. The method of claim 2, wherein said silage comprises at least one of animal or vegetable matter.

4. The method of claim 3, wherein said animal matter comprises at least one of fish, poultry, or cattle.

5. The method of claim 3, wherein said vegetable matter comprises at least one of grass, legume, whole-crop cereal, potato, or food manufacture waste.

6. The method of claim 5, wherein said grass is selected from rye grass, timothy, cocksfoot, or fescue.

7. The method of claim 5, wherein said legume is selected from alfalfa, pea, clover, lucerne, sainfoin, field bean, common vetch, or lupin.

8. The method of claim 5, wherein said whole-crop cereal is selected from corn, sorghum, barley, wheat, oats, or rye.

9. The method of claim 5, wherein said food manufacture waste is selected from sugar cane, sugar beets, peas, or fruit waste.

10. The method of claim 2, wherein said high-moisture grain is selected from corn, sorghum, barley, wheat, oats, or rye.

11. The method of claim 1, wherein said treating comprises applying said Lactobacillus plantarum ATCC
53187 or mutants thereof to said agricultural products by mixing or spraying.

12. The method of claim 11, wherein said mixing or spraying uses a bacterial composition comprising Lactobacillus plantarum ATCC 53187 or effective mutants thereof.

13. The method of claim 12, wherein said bac-terial composition is in a liquid or dried form.

14. The method of claim 12, wherein said composition comprises additional bacterial strains.

15. A method of suppressing the growth of fungi in an agricultural product which comprises treating said product with an effective amount of Lactobacillus plantarum ATCC 53187 or effective mutants thereof.

16. A biologically pure culture of an organism selected from the group consisting of:
(a) Lactobacillus plantarum ATCC 53187 which possesses a fungal inhibiting property, and (b) a mutant derivative of Lactobacillus plantarum ATCC
53187 which possesses a fungal inhibiting property that is equivalent to said fungal inhibiting property of Lactobacillus plantarum ATCC 53187;
wherein said culture is capable of use in enhancing the preser-vation of an agricultural product.

17. A composition of matter comprising the biologically pure culture of claim 16 and a carrier.