WO1993002558A1

WO1993002558A1 - Method and formulation for reducing microbial populations

Info

Publication number: WO1993002558A1
Application number: PCT/US1991/003796
Authority: WO
Inventors: Ivan A. Casas-Perez
Original assignee: Biogaia Biologics Ab
Priority date: 1991-08-01
Filing date: 1991-08-01
Publication date: 1993-02-18

Abstract

A formulation including one or more pure cultures of Lactobacillus, such as L.reuteri, L.animalis and L.salivarius and a sugar source, such as whey and a method of feeding animals which utilizes the formulation to be ingested by the animals with their normal food.

Description

METHOD AND FORMULATION FOR REDUCING MICROBIAL POPULATIONS

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates generally to a method and composition for reducing microbial populations, and more particularly, to a composition containing Lactobacillus and a carbohydrate for use in decreasing the numbers of pathogens in the gastrointestinal tract which do not utilize lactose, such as Salmonella.

Description of the Related Art

Lactobacillus reuteri, L. animalis. and L. salivarius are naturally occurring microorganisms in the GI tract of animals including domestic avian species (Sarra, et al., 1985, System Appl. Microbiol 6:86-89). This paper and all other papers cited herein are hereby incorporated herein by reference.

As disclosed in co-pending application Serial No. 07/268,361 filed September 19, 1988, the disclosure of which is incorporated herein, Lactobacillus reuteri is a symbiotic resident of the gastrointestinal (GI) tracts of humans, swine and other animals. The neotype strain of L. reuteri is DSM 20016 (ATCC No. 53609). This strain and strain 1063 (ATCC No. 53608), discussed in the co-pending application, are available to the public at the American Type Culture Collection (Rockville, MD) having been deposited therein April 17, 1987. The GI tract of animals is a complex ecosystem harboring an estimated 300-500 species of microorganisms, known collectively as the indigenous microbiota or microflora. Despite over 100 years of intensive research in the field of intestinal microbiology much remains to be learned about these microorganisms, the complex interrelationships that exist between the different species, and the nature of the symbiotic relationships existent between the microbiota and their host.

Under certain conditions some members of the indigenous microbiota can become opportunistic pathogens causing a variety of enteric diseases. More often, however, pathogens gain access to the GI tract as contaminants in food or water. Notable among the latter . are a number of bacterial genera including Escherichia. Salmonella, Shiσella, ϊersina. Vibrio, Campylobacter and Clostridiurrt- as well as viruses (e.g., roto-, astro- and ciliciviruses) and intestinal parasites (e.g., Giardia and Entamoeba species) . Acute and chronic enteric diseases caused by these and other microorganisms occur worldwide causing considerable human misery and loss of economically important animals. Certain microbial activities have also been associated with production of mutagens within the GI trac .

It is also known that other members of the indigenous microbiota exist in a symbiotic or synergistic relationship with their host contributing in many positive ways to the host's general health and well-being. It is well-known that germ-free animals are very susceptible to pathogens and have poorly developed GI tracts. In return for the nutrient-rich and stable ecosystem provided for them, the indigenous microbiota can provide their hosts with an assortment of benefits including among others protection against enteric pathogens (a process known as colonization resistance or competitive exclusion) , stimulation of normal development and function of the GI mucosa, production of various vitamins and other nutrients, and re-metabolism of the host's abundant endogenous mucosal tissue. It has been reported on numerous occasions that the enteric lactobacilli (i.e., bacteria belonging to the genus Lactobacillus which reside in the GI tract and which include a large number of nonpathogenic, non-toxic bacteria) play an important role in the health and well- being of their human and animal hosts. Included in this role is their ability to regulate conditions in the GI tract so that the different populations of microorganisms in this system are kept in a healthy balanced state (Fuller, 1977, Br. Poultry Sci. 18:85-94). Certain Lactobacillus species in fact are added to human and animal foodstuffs either to preserve them, enhance their flavors and/or exert other beneficial effects in the GI tract. Lactobacillus plantarum strains, for example, are grown commercially in large amounts and used as starter cultures for the commercial preservation of a variety of human foods (meats, vegetables, and dairy products) and animal foods (silage). Lactobacillus acidophilus strains are grown commercially in large amounts to be added to human (e.g., milk) or animal (feedstuffs) foods as a means of introducing these bacteria into the GI tract where they can exert beneficial effects. Although these bacteria are likely to be already present in the GI tract their numbers may vary widely from individual to individual, and therefore beneficial effects of these bacteria may not be present in persons deficient in these bacteria. Reports on the beneficial effects resulting from the oral administration of live Lactobacillus cultures have increased in recent years with findings that dietary Lactobacillus therapy affords protection from colon cancer for human populations on western diets, reduces the incidence of experimentally induced large bowel tumors in rats, reduces the fecal concentration of bacterial enzymes known to catalyze the conversion of procarcinogens to proximal carcinogens in humans, and reduces the serum cholesterol levels in swine.

Themetabolic endproducts ofLactobacillus metabolism, such as acetic acid, lactic acid and hydrogen peroxide, are well-known for their antimicrobial activities. They are believed to play a significant role in maintaining proper conditions within the GI tract. Some lactobacilli produce bacteriocins or bacteriocin-like proteins which also exhibit bacteriocidal activity toward other members of that species or closely related species. Reports have appeared concerning low molecular weight, antimicrobial substances produced by lactobacilli. With the exception of reuterin which is produced by Lactobacillus reuteri (copending application) none of these low molecular weight substances has been identified and these reports have not been confirmed. In fact, some of these substances have proven to be none other than lactic acid, acetic acid or hydrogen peroxide.

Several studies have been conducted to determine the effect of lactobacilli on the performance of domestic avian species. Some of these studies indicate that dosing broilers with L. acidophilus improved their growth (Tortuero, 1973, Poultry Sci. 52:197-203, and Dilworth and Day, 1978, Poultry Sci. 57:1101). An increase in egg production as a result of addition of lactobacilli to laying hens feed has also been reported (Hargis and Creger, 1978, Poultry Sci. 57:1103). Improvement of young turkeys' body weight and feed efficiency was obtained with a Lactobacillus product added to the feed (Francis et al., 1978, Poultry Sci. 57:1687- 1689). Similar effects were reported (Potter et al., 1986, Poultry Sci. (Suppl. 1) 65:107) when L. acidophilus in combination with varying protein levels was fed to turkeys up to 12 weeks of age, but no difference with the controls was observed at 16 weeks. Dosage of 10⁷ colony forming units (CFU) and higher depressed chick growth (Watkins and Kratzer, 1982, Poultry Sci. 61:1565, and, 1984, Poultry Sci. 63:1671-1673).

Other strains of lactobacilli did not stimulate broilers weight gain (Watkins and Kratzer, 1984), and did not stimulate egg production (Goodling et al., 1987, Poultry Sci. 66:480-485). Damron et al. (1981, Poultry Sci. 60:1350-1351) did not find any beneficial effect of L. acidophilus and other lactobacilli cultures in turkey breeder hens. Nurmi and Rantala (1973, Nature 241:210-211) demonstrated that the intestinal microflora present in some adult chickens (i.e. the cecal microflora) interferes with colonization by salmonellae of newly hatched chicks. The application of this concept, known as the Nurmi concept or competitive exclusion, has been successfully tested in some laboratories and is also used commercially (Watkins and Miller, 1983, Poultry Sci. 62:1772-1779; Wierup et al., 1988, Poultry Sci. 67:1026-1033; Impey et al., 1982, J. Hyg. Camb. 89:749; Snoeyembos et al., 1978, Avian Dis. 22:273-287). There are many problems associated with this method, in particular, a lack of adequate selective isolation and characterization techniques to study and consistently obtain cecal flora preparations (Bailey, 1988, Poultry Sci. 67:928-932; Mead and Impey, 1986, J. Bacteriol. Symp. Suppl. 675-755; Soerjadi et al., 1981, Avian Dis. 25:1027-1033.

Mannose and lactose were shown to significantly reduce Salmonella typhimurium adherence to the ceca of chicks (Oyofo et al., 1989, Avian Dis. 33:531-534). The inhibitory effect of these sugars was believed to take place by blocking the receptor sites on the gut epithelium and on the microorganism pili. It has been shown that providing dietary lactose together with cecal flora contents to broiler chickens reduced the occurrence of Salmonella (Corrier et al., 1990, Avian Dis. (in press); Hinton et al., 1990, Avian Dis. (in press); Oyofo et al., 1989) . Historically, Lactobacillus administration (i.e., inclusion of viable cells in the feed) to animals has not yielded consistent benefits. There are many reasons for this including, for example, using Lactobacillus species or strains unadapted to or unsuitable for the animal being treated, or using conditions which do not produce a colonization of the Lactobacillus within the GI tract.

The object of this invention is to provide a food or feed additive formulation and method comprising isolated and identified pure cultures of Lactobacillus reuteri and/or other Lactobacillus species together with a sugar source such as lactose, using whey as a source for this sugar.

Lactobacillus reuteri was previously found to be recoverable from the stools of piglets after the piglets had been fed large quantities of the cells (co-pending application) . It was hypothesized in the co-pending application that such cells could be used, possibly in conjunction with glycerol, as a growth and health aid for these animals. Evidence has been obtained (and presented below) that L.reuteri may be administered to poultry as a means of competitively excluding salmonellae.

It is an object of this invention to provide a formulation that may be used as a food or food additive ixture that utilizes pure cultures of one or more of certain Lactobacillus species in addition to a sugar source.

It is a further object of the invention to provide a formulation that results in rapid weight gain for growing animals.

It is a further object of the invention to provide a formulation that decreases the number of pathogenic microorganisms in the gastrointestinal tract, with the purpose of adding any sugar for at least the purpose of being a source of carbohydrate for the metabolism of the Lactobacillus but not utilized by the animal or the unwanted microorganism(s) .

Other objects and advantages will be more fully apparent from the following disclosure and appended claims.

SUMMARY OF THE INVENTION The invention is for a formulated product that may be used as an animal feed additive and that includes isolated and identified pure culture(s) of naturally occurring gastrointestinalmicroorganisms, for example, Lactobacillus reuteri, L. animalis, and/or L. salivarius. The formulated product also includes a sugar source. The invention also includes a method of feeding the formulation to animals. Preferably the sugar source is whey, when animals which do not metabolize lactose such as chickens are used, because whey contains the sugar lactose and is an easily obtainable and voluminous waste product. The formulation of the invention when fed to animals provides a means to decrease populations of undesirable gastrointestinal microbes and results in increased weight gain of the animals, especially under the less than optimum growth conditions normally present in commercial livestock environments.

Other aspects and features of the invention will be more fully apparent from the following disclosure and appended claims.

DETAILED DESCRIPTION OF THE INVENTION AND

PREFERRED EMBODIMENTS THEREOF The present invention provides a formulation usable as a food or feed additive for animals. In the particular invention, such animals specifically include all poultry and mammals, including human beings. The formulation for a particular animal comprises one or more pure cultures of a Lactobacillus species naturally occurring in the gastrointestinal tract of that animal and a source of a sugar that is metabolizable by the Lactobacillus species but not to any great extent by the animal. Thus, the formulation of the invention comprises:

(a) a bacterial culture comprising at least one live pure culture of a Lactobacillus species which occurs naturally in a particular animal group; and

(b) a source of sugar metabolizable by the Lactobacillus species in the bacterial culture but not metabolizable by the animals in the grou . By the term "group" is meant animals of a particular species or group of species which share in common a tendency to have a similar gastrointestinal Lactobacillus flora and a similar inability to metabolize a sugar which is metabolizable by the Lactobacillus flora. As discussed below, the formulation discussed in detail herein has been devised for poultry but is adaptable to other animals, and includes a source of lactose which is not metabolizable by poultry.

In the preferred embodiment, the formulation comprises live, pure cultures of at least one of Lactobacillus reuteri, L. animalis and L. salivarius, and a sugar source. The preferred sugar source is whey, because it is inexpensive and easily available, and because it contains lactose, a good source of carbon and energy for growth of the added microorganisms. An additional advantage of using a lactose source for feeding poultry or other birds is that birds do not utilize this sugar, and it is therefore readily available for the added microorganisms. Preferably, powdered whey is utilized as the lactose source to minimize shipping costs and spoilage prior to formulation of the additive. The preferred method of formulating the additive is as follows. L.reuteri. L.animalis and L.salivarius are grown individually in a variety of appropriate media used for lactobacilli. Lactose or maltose are the preferred sources of energy so that the cells are capable of rapid metabolism of the carbohydrates which may be present in the formulation or in the animals' food. The cells used for the preparation of the additive may be freshly harvested, frozen, lyophilized or suspended in oil or a specifically formulated diluent such as an aqueous solution. Commercially available whey powder or whey concentrate is used to formulate the additive. Although the cells and whey may be fed separately, the are preferably mixed together with or without other ingredients (e.g. corn, soybean meal, wheat, etc.). The mixture may be of a variety of microbe and whey mixtures, for example a solid and a solid (e.g. fine powder with granulated whey, etc.), a liquid and a solid (cell suspension and whey), a solid and a liquid (lyophilized cells and. a liquid whey concentrate) or a liquid and a liquid (liquid cell suspension and a liquid whey concentrate) . The additive final presentation of the mixture could be as a powder, granules, or pellets or liquid. Animals may be fed the additive in a variety of ways: for example, (1) the additive may be combined with dry feed during feed milling or when the feed is delivered to the animals; (2) the additive may be sprinkled on the food as a powder; or (3) the additive may be mixed in the drinking water. Preferably, to minimize labor, the additive is mixed with dry feed.

The features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be considered as limiting the invention.

EXAMPLES Example l. Growth of Turkey Poults to be Fed Additive Experimentally

One day old Nicholas turkey torn poults are used in this study. The poults are not toe clipped, desnooded or wing clipped, nor are they given any vaccinations.

The turkeys are placed in animal rooms at the Dearstyne Poultry Research Center, Department of Poultry Science at NCSU's Agricultural Research Service (NCARS). The animal rooms have controlled ambient temperature, day length and thermostatically controlled Petersime brooding batteries (Petersime Incubator Co., Petersime, OH). A normal turkey starter diet, for example as shown in Table 1 with and without whey powder, is used throughout the trial. The amount of whey in the diet allows for a final 2.2% lactose. The trial is twenty days in duration, covering the period from day of hatch to day 12. The turkeys are weighed on Day 0 (at hatch), Day 5, Day 12, Day 15 and Day 20.

turkeys, are the strains which are used. The Lactobacillus strains are grown in LCM medium utilizing lactose or maltose for 24 h at 37°C, harvested by centrifugation, and washed twice with fresh basal medium as previously described (Axelsson et al., 1989, Microbial Ecol. Health Dis. 2:131-136; Chung et al., 1989, Microbial Ecol. Health Dis. 2:137-144). These cells are mixed into the animal feed at a level of approximately 10⁵ CFU g'¹ of feed. This inoculum level has been shown to effectively enhance the population level of this microorganism in the chicken ceca (Casas et al., 1990, in preparation). The number of L♦ reuteri in the feed and in the ceca are monitored as previously described (Chung et al., 1989, Microbiol. 56: 943-948). Appropriate dilutions are plated onto LBS agar and incubated anaerobically (Gas-Pak jars) at 37°C for 48 h. Plates containing about 50 to 200 CFU are overlaid with glycerol agar seeded with L. plantarum indicator cells, reincubated anaerobically for 24 h, and colonies showing growth inhibition zones counted as reute in-producing L.reuteri cells.

S. senf enberσ, isolated from turkeys, resistant to novobiocin and nalidixic acid, was obtained from Evillmar

Poultry Co. (Evillmar, MN) . Inocula for infectious challenge are prepared from cultures grown in BHI Broth (Difco Laboratories, Inc., Detroit, MI) and incubated at

37°C for 24 hours. The cultures are diluted appropriately in sterile 50 mM phosphate buffer, pH 7.0, to obtain challenge inocula containing 10⁶ CFU per ml. Enumeration of these Salmonella is carried out by plating appropriate dilutions on Rembach Salmonella medium (Rembach, 1990,

Appl. Environ. Microbiol. 56:301-303).

Caecal content samples formicrobiological enumeration are prepared from sacrificed birds. Caeca are carefully removed from the birds and the open end of each is clipped. The exterior of the caecum is alcohol sterilized before transferring its contents to a stomacher bag for mixing and further dilution.

Example 3. Treatment of Poults

Turkey poults of Example 1 are subjected to the following eight treatments with two pens of 15 birds per pen being in each group:

Salmonella senftenberg infected group 1. Control, no whey, no L.reuteri

2. No whey, L.reuteri

3. Whey, no L.reuteri

4. Whey, L.reuteri

L.reuteri, when administered is mixed into the feed. The inoculated feed is changed every two days to guarantee the presence of viable L. reuteri in the feed. Whey is added to the feed, before milling, for a final 5% lactose concentration. Alternatively, L. reuteri and concentrated or dehydrated whey are formed into tablets , or added together in any product in which both components in a liquid or solid form have been previously combined, and the combination added to the feed of the animals.

5. senftenberg (10⁶ CFU per ml) is crop fed by the means of an animal feeding stainless steel needle attached to a hypodermic syringe on day 5 after hatch. Example 4. Results of Addition of Lactobacillus, Whey and Salmonella

Salmonella senftenberg in feces and caecal contents of poults treated as in Example 3 is shown in Tables 2 and 3, respectively. The effect of L. reuteri and whey on the number of S. senftenberg in feces (droppings) becomes obvious at 72 h after Salmonella challenge. The data indicate a synergistic effect when whey and L. reuteri are added together.

The presence of S. senftenberg in caecal contents is presented in Table 3. The results show that addition of L. reuteri and/or whey, but in particular, the combination of . reuteri and whey, is effective in reducing the presence of S. senf enberg in the ceca of these animals. Thus, whereas 47% of the control ceca tested positive for S. senftenberg, none (0%) of the samples tested positive when fed L. reuteri and whey.

TABLE 2. Log₁₀ CFU of S. senftenberg per g feces 72 h post challenge.

TABLE 3. Percent of cecal samples testing positive for S. senftenberg and L. reuteri 7 days post challenge

¹ Positive samples had > 10⁷ CFU/g

² Positive samples had < 10³ CFU/g

Example 5. Growth of Cold Stressed Poults Fed with L. reuteri and Whey

Instead of exposing turkey poults to constant temperature rooms as in Example 1, the temperature in the pens of cold stressed birds is 90 degrees F for 1 hour, then 85 degrees G for 2 hours in an on-off cycling for 48 hours after hatch. The temperature is then set back to normal brooding temperature for the remainder of the experiment, normal brooding temperature being 90 degrees F for the first seven days after hatch, 85 degrees F from day 7 to day 10, and 75 to 80 degrees after day 10.

Turkey poults are subjected to the following four treatments, with eight pens of eight birds per pen being in each treatment group:

1. Control, no whey, no L. reuteri 2. No whey, L. reuteri 3. Whey, no L. reuteri

4. Whey, L. reuteri

Example 6. Results of Growth of Cold Stressed Poults

Relative wights of poults treated as in Example 5 , at 0, 5, 10, 15, and 20 days of age are shown in Table 4. The beneficial effect of whey becomes evident at day 5, while the effect of L. reuteri becomes obvious at days 15 and 20.

TABLE 4. Effect of L. reuteri and whey on body weight of turkey poults.

TREATMENTS RELATIVE WEIGHT (PERCENT) AT

Day 0 Day 5 Day 10 Day 15 Day 20

For percentage conversion, control weights (no whey, no L. reuteri) at each weight day were made equal to 100%.

Example 1. Use of Lactobacillus salivarius and

Lactobacillus animalis L. salivarius subsp. salivarius ATCC type strain No. 11741 and L. animalis ATCC type strain No. 35046 are grown as in Example 2. Each strain is added individually to feed as in Example 3. The feed is augmented with whey according to Example 3. The feed is used to feed chickens and turkeys to decrease undesirable microbial organisms and improve poultry weight gain.

Example 8. Use of Multiple Lactobacillus Strains

Strains of the three Lactobacillus species discussed in Examples 3 and 8 are each added individually, or as a mixed inoculum to the whey-augmented feed according to Example 3. The feed containing the three strains is used to feed turkeys and chickens.

BEST MODE FOR CARRRYING OUT THE INVENTION

The food or feed additive for animals, such as poultry and mammals, including human beings, comprises one or more pure cultures of a Lactobacillus species naturally occurring in the gastrointestinal tract of that animal and a source of a sugar that is metabolizable by the Lactobacillus species but not to any great extent by the animal. Preferably, the formulation comprises live, pure cultures of at least one of Lactobacillus reuteri. L. animalis and L. salivarius, and a sugar source. The preferred sugar source is whey, particularly for poultry. Whey is inexpensive and easily available, contains lactose, a good source of carbon and energy for growth of the added microorganisms, and is not utilized poultry or other birds and therefore readily available for the added microorganisms. Preferably, powdered whey is utilized as the lactose source to minimize shipping costs and spoilage prior to formulation of the additive.

The preferred method of formulating the additive is as follows. L.reuteri, L.animalis and L.salivarius are grown individually in a variety of appropriate media used for lactobacill. The cells used for the preparation of the additive may be freshly harvested, frozen, lyophilized or suspended in oil or a specifically formulated diluent such as an aqueous solution. Commercially available whey powder or whey concentrate is used to formulate the additive. The cells and whey are preferably mixed together with or without other ingredients (e.g. corn, soybean meal, wheat, etc.). Animals are preferably fed the additive as a mixture with dry feed.

INDUSTRIAL APPLICABILITY The invented formulation may be used as a food or food additive mixture for animals grown commericially, such as poultry. It provides a product that results in rapid weight gain for growing animals, and decreases the number of pathogenic microorganisms in the gastrointestinal tract, thereby resulting in increased productivity and decreased animal mortality.

While the invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the invention.

Claims

THE CLAIMSWhat Is Claimed Is:

1. A formulation for oral administration to an animal of a particular group, comprising: (a) a bacterial culture comprising at least one live pure culture of at least one Lactobacillus species which occurs naturally in a particular animal group; and (b) a source of sugar metabolizable by the Lactobacillus species in the bacterial culture but not metabolizable by the animals in the group.

2. A formulation for oral administration according to Claim 1, comprising a Lactobacillus culture selected from the group consisting of L.reuteri, L.salivarius and L.animalis, and the animals are poultry.

3. A formulation for oral administration according to Claim 1, wherein the source of sugar comprises whey.

4. A formulation for oral administration according to Claim 1, wherein the Lactobacillus culture is selected from the group consisting of L.reuteri, L.salivarius and L.animalis, and the source of sugar is whey.

5. A formulation for oral administration according to Claim 4, further comprising glycerol.

6. A formulation for oral administration according to Claim 1, wherein pure cultures of L.reuteri. L.salivarius and L.animalis are used, and the sugar is lactose.

7. A formulation for oral administration according to Claim 6, wherein the source of lactose is whey.

8. A formulation for oral administration to animals, comprising: (a) a bacterial culture consisting of at least one live, pure culture of Lactobacillus: and (b) a source of sugar.

9. A formulation for oral administration according to Claim 8, comprising a Lactobacillus culture selected from the group consisting of L.reuteri. L.salivarius and L.animalis. and the animals are poultry.

10. A formulation for oral administration according to Claim 8, wherein the source of sugar comprises whey.

11. A formulation for oral administration according to Claim 8, wherein the Lactobacillus culture is selected from the group consisting of L.reuteri, L.salivarius and L.animalis. and the source of sugar is whey.

12. A formulation for oral administration according to Claim 11, further comprising glycerol.

13. A formulation for oral administration according to Claim 8, wherein pure cultures of L.reuteri. L.salivarius and L.animalis are used, and the sugar is lactose.

14. A formulation for oral administration according to Claim 13, wherein the source of lactose is whey.

15. A method of decreasing numbers of undesirable microbes in an animal's gastrointestinal tract, comprising: (a) obtaining at least one pure culture of Lactobacillus cells;

(b) obtaining a source of sugar; and

(c) administering the Lactobacillus cells and the sugar orally to the animal.

16. A method of decreasing numbers of undesirable microbes in gastrointestinal tracts of animals, comprising:

(a) obtaining at least one pure culture of Lactobacillus cells;

(b) obtaining a source of sugar metabolizable by the Lactobacillus cells and not to a significant extent by the animals or the undesirable microbes; and

(c) administering the Lactobacillus cells and the sugar orally to the animal.