WO1997010801A2

WO1997010801A2 - Nisin compositions to prevent the promotion of tooth decay by suppressing formation of acid from foods by oral bacteria

Info

Publication number: WO1997010801A2
Application number: PCT/US1996/015073
Authority: WO
Inventors: Peter Blackburn; Beth P. Goldstein
Original assignee: Ambi Inc.
Priority date: 1995-09-20
Filing date: 1996-09-19
Publication date: 1997-03-27
Also published as: ZA967950B; AU7365596A; WO1997010801A3

Abstract

The invention concerns a method for preventing or suppressing the fermentation by oral bacteria of sugars per se as well as sugars produced by breakdown of complex carbohydrates by salivary enzymes, which employs a lathionine-containing bacteriocin as active agent. The bacteriocin is present in an amount sufficient to inhibit fermentation without exhibiting a significant effect on bacterial viability. The bacteriocin may be employed as an additive to sugar- and starch-based foods, confectionieries and beverages or may be incorporated into a formulation for use following consumption of such products.

Description

NISIN COMPOSITIONS TO PREVENT THE PROMOTION

OF TOOTH DECAY BY SUPPRESSING FORMATION OF

ACID FROM FOODS BY ORAL BACTERIA

BACKGROUND OF THE INVENTION Nisin is a polypeptide with antimicrobial properties and is produced in nature by various strains of the bacterium Lactococcus (Streptococcus) lactis. It is a known food preservative which inhibits the outgrowth of spores of certain species of Gram-positive bacilli. Nisin is a preservative found naturally-occurring in low concentrations in milk and cheese, and is believed to be completely nontoxic and nonallergenic to humans. Since nisin is a polypeptide, any residues remaining in foods are quickly digested upon reaching the intestine.

Recently, compositions comprising nisin, particularly in combination with various non-bactericidal agents, were shown to be highly active against various species of Gram-positive and Gram-negative bacteria (U.S. Pat. Nos. 5,135,910; 5,217,950 and 5,260,271). The antibacterial activity of nisin and indeed of other chemically related bacteriocins is generally described by a rapid and extensive bactericidal action against the target bacteria.

Nisin has been recognized as safe by the FDA as a direct food ingredient in pasteurized cheese spreads and pasteurized, processed cheese spreads which may or may not also include fruits, vegetables or meats. The FDA-approved nisin preparations are specified as having a nisin content of not less than 900 international units per milligram and having a quantity of active ingredient that delivers a maximum of 250 ppm of nisin in the finished product. The preparations are specified for use in inhibiting the outgrowth of Clostridium botulinum spores and toxin formulation in the above mentioned spreads (see 21 C.F.R. § 184.1538). Nisin has also been recognized as useful in the inhibition of growth of Listeria in such foods. However, the use of nisin for the inhibition of sugar fermentation by oral microflora at dosages which are sublethal for the microflora found in the oral cavity has not previously been contemplated.

Acid, produced from bacterial fermentation of sugars and other carbohydrates in the mouth, causes partial dissolution of tooth enamel, providing a focus for further bacterial growth, plaque formation and decay. Consumption of sweet foods (particularly candy) and beverages is clearly correlated with development of tooth decay, presumably because these foods and beverages have a high content of readily fermentable sugars. Therefore, suppression of bacterial fermentation in the mouth would be expected to be of particular benefit to people consuming these foods. However, fermentable sugars and other carbohydrates, particularly complex carbohydrates such as sucrose, lactose or starch which can be converted to simple fermentable sugars, are present in a wide variety of other foods, and it is thus likely that some individuals could benefit from a wider application of the suppression of bacterial fermentation in the mouth.

It is thought that Streptococcus mutans is the principle oral bacterium responsible for the acidogenesis that leads to tooth decay. However, the oral microflora of humans comprises many other bacterial species, some of which are thought to contribute to acid formation and tooth decay and some of which are normally present in a healthy mouth. Among the species of bacteria which may be present in the oral cavity and against which nisin is effective are Streptococcus sanquis. Streptococcus mutans. Streptococcus milleri. Streptococcus mitis, Streptococcus mitior. Streptococcus salivarius. Streptococcus pyoqenes. Staphylococcus aureus. Gardnerella vaqinalis. Lactobacillus odontolvticus, Actinomvces odontolyticus. Actino yces israelli. Actino yces naeslundi. Actinomvces actino vcetescomitans. Fusobacterium nucleatum. Bacteroides intermedius. Peptostreptococcus micros. Porphyromonas inqivalis. Bacteroides ureolvticus and Wolinella recta. Drastic reduction of the normal oral flora (as sometimes occurs, for example, upon prolonged antibiotic therapy for infectious diseases) may lead to the overgrowth of pathogenic bacteria or yeasts. It would therefore be of potential benefit to be able to suppress fermentation of sugar and other carbohydrates such as starch during, and for a short time after, consumption of foods, without at the same time causing significant reductions in the resident populations of bacteria, so as to reduce or inhibit the potential of carbohydrates to promote tooth decay.

SUMMARY OF THE INVENTION

It has now been found that low concentrations of nisin which do not significantly reduce the numbers of viable bacteria, nevertheless can inhibit the ability of the natural mixed population of oral bacteria to ferment sugars. The invention provides bacteriocin compositions of nisin or other lanthionine-containing bacteriocins, as well as compositions in which bacteriocins are combined with various non-antibacterial agents, for example chelating agents or surfactants. The invention further provides the compositions dissolved or suspended in foods, gums, confectionery or beverages containing fermentable sugars or complex carbohydrates, such as sucrose, lactose or starch, capable of converting to simple fermentable sugars. The invention also provides the compositions present as surface coatings or dustings of the above foodstuffs. Finally, the invention provides for formulations for use immediately following consumption of carbohydrate-containing foods and beverages.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1D show the effect on the pH of saliva in vitro due to the fermentation of various sugars by the native microbial flora.

Figures 2A-2C show the effect in vitro of varying concentrations of nisin on the fermentation of glucose, sucrose and fructose, respectively, in saliva.

Figure 3 shows the effect in vitro of varying concentrations of nisin on glucose fermentation in saliva.

Figure 4 shows the effect on glucose fermentation of a fixed concentration of nisin introduced directly into the mouth. Figures 5A and 5B show the effect in vitro of the absence and presence, respectively, of citrate on glucose fermentation in saliva in the presence of nisin.

DETAILED DESCRIPTION OF THE INVENTION We have found that nisin, when added to human saliva containing native salivary bacteria and a simple or complex fermentable sugar (such as glucose, fructose or sucrose) , inhibits the acidification of the saliva which otherwise occurs due to the action of the bacteria on the sugar. Contrary to what might be expected, this inhibition was found to occur at nisin concentrations that were too low to produce the rapid, extensive cell killing that, in the prior art, has been associated with the action of nisin and other related bacteriocins on bacteria. Compositions containing these low concentrations of nisin are useful in foodstuffs, beverages, etc. , which contain simple or complex fermentable sugar or in other orally administered compositions as a method of protecting against tooth decay.

According to the invention, nisin at concentrations between 1 and 5 μg/ml significantly retarded acidification caused by the bacterial fermentation of sugar, and nisin concentrations between 10 and 30 μg/ml totally suppressed the acidification as monitored by pH drop over a 6 h observation period. It was noteworthy that nisin concentrations of up to 10 μg/ml produced only very slight decreases (less than one log₁₀) in the apparent numbers of viable bacteria in the saliva, and this effect was transient, with recovery taking place after the initial apparent drop in titer. Even at 30 μg/ml of nisin, only a transient drop in bacterial titer (of up to slightly more than one log₁₀) was observed, although the recovery period was somewhat longer than at lower concentrations of nisin.

Low concentrations of nisin and other lanthionine- containing bacteriocins are particularly suited to reducing or suppressing acid production from bacterial fermentation of sugar and other carbohydrates present in foods and beverages. Potential food-related uses include addition to foods, in particular those with high sugar content, such as candies, beverages (e.g. juices and sodas) , chewing gums, etc. Starch is readily converted by salivary enzymes to fermentable sugars, and thus the bacteriocin compositions of the invention would also have use as additives to starchy foodstuffs. Even in cases of foods wherein artificial sweeteners are used, there is no substitute for starch, and many artificially sweetened products can be promoters of tooth decay by virtue of their starch content. The bacteriocin compositions, then, may in general also be added to foods with a high content of starch, whether or not they have a high sugar content, for example, cold cereals; yogurts; prepared puddings; ice creams, frozen desserts, cookies and cakes; and chewy candies such as jelly beans and licorice.

The low, non-bactericidal concentrations of nisin according to the invention can be expected to be effective as an additive to any food or beverage the preparation of which does not compromise the integrity of the active agent. In cases of foodstuffs wherein preparation conditions might reduce the efficacy of prior-added bacteriocin, it is envisioned that compositions according to the invention can be added as components of, for example, condiments to such foods as potatoes and pastas, for example, catsup or spaghetti sauce. As a further example, the compositions can be added in the form of a glaze or powdered coating to the surface of baked goods. In addition, the bacteriocin can be added to the foodstuff in sufficient excess, such that the residual or available bacteriocin following processing of the foodstuff is present at a concentration capable of suppressing acidogenesis in the mouth. In addition to those products mentioned above, such products include, but are not limited to, infant formula; sweet prepared foods; breads; canned, jarred or frozen vegetables with relatively high sugar content (e.g., corn niblets, beets, carrots and sweet potatoes) ; sweet salad dressings; sweet toppings or dips; ice cream; prepared puddings; sweetened whipped cream or nondairy whip; sweetened salads; canned fruits, fruit salads and fruit toppings; prepared chocolate milk and other flavored milk preparations; sweetened or fruit- flavored yogurts; processed cheeses, particularly those containing fruits, vegetables and nuts; and sweet sauces such as sweet and sour sauce, cocktail sauce and catsup.

Further uses of the composition include incorporation into toothpastes, mouthrinses, sprays, gums, or lozenges designed to be used immediately after the consumption of foods and beverages in order to prevent acid formation. This aspect of the invention would be expected to have applicability in connection with the consumption of virtually any food or beverage containing fermentable sugar and/or complex carbohydrate convertible to fermentable sugar.

Nisin in combination with chelators and mild surfactants has been found to have enhanced bactericidal activity, see for example, U.S. Patent Nos. 5,135,910; 5,217,950 and 5,260,271, the disclosures of whose specifications are hereby incorporated by reference in their entirety. It is envisioned that such combinations in very low, sublethal amounts will also be effective in the suppression or prevention of sugar and other carbohydrate fermentation by the microbial flora of the oral cavity.

Nisin and other related peptide bacteriocins are considered to be contact bactericides, rapidly killing at least 99.9% of susceptible bacteria, and this in fact is their mechanism of action under previously tested conditions at concentrations previously judged to be efficacious in the oral cavity. Thus the effect of sub-bactericidal concentrations of nisin in suppressing bacterial fermentation is surprising. In particular, the fairly broad 'window' between concentrations that retard acid formation and concentrations that are strongly bactericidal is quite unexpected.

In order to demonstrate these newly observed properties of nisin, a number of experiments were conducted, most of them in fresh human saliva. The test conditions described herein used a 'natural' bioload (fresh human saliva) and the results are therefore believed to be predictive of what would happen in saliva and plaque in the human mouth with respect to inhibition of fermentation of sugars and other carbohydrates such as starch. These experiments are meant as illustration and are not intended to limit this invention. It is to be expected that other lanthionine-containing peptide bacteriocins would be effective substitutes for nisin and that employment of the bacteriocins according to the claimed methods would be effective in inhibiting fermentation not only of sugars directly present in foods but also sugars derived from breakdown by salivary enzymes of starch and other complex carbohydrates that can convert to fermentable sugars.

The data from the tests described below indicate that nisin over a defined range of concentrations can inhibit fermentation of sugars by oral bacteria without compromising the viability of the bacteria. However, one of skill in the art would recognize that in the context of the present invention, such factors as dilution, interaction of the active agent with its surrounding components and the effects of processing on the availability of bacteriocin in a foodstuff should be taken into account during the preparation of candies, food, and beverages. Therefore, the compositions and methods of the present invention contain and employ concentrations of the active agent which will provide effective amounts of the bacteriocin sufficient to inhibit bacterial fermentation of sugars in the mouth without compromising the viability of the bacteria. This effect of the active agent would be expected to decrease the incidence and severity of tooth decay. Considering the factors mentioned above, the concentration of bacteriocin required to achieve the effect according to the invention is expected to be from about 0.1 μg/ml to about 100 μg/ml in a beverage or from about 0.1 ppm to about 100 ppm in a solid food, as appropriate. It is expected that actual delivered concentrations to the site of action in the range from about 0.1 μg/ml to 30 μg/ml of fluid in the oral cavity or 0.1 to 30 ppm concentration in the oral cavity would be effective.

All tests in the following examples were conducted at 37'C. The raw material for these experiments consisted of diluted (approximately two-fold) human saliva, collected after rinsing the mouth for a brief period of time (generally 30 seconds) with small volumes (generally 2 ml) of liquid. In some experiments, individual rinses (with water or with 5% w/v sugar solutions, with or without nisin) were collected over a period of time. In other experiments, a pool of saliva was constituted by combining several rinses of sugar solution collected over a short period of time (about 15 min) ; the homogeneous pool was then subdivided for exposure to different concentrations of nisin. Saliva samples were incubated aerobically. In the various examples, parameters measured included: initial pH, pH change over time, and bacterial count (CFU/ml) determined by standard dilution and plating techniques on tryptic soy agar containing 5% sheep blood. Plates were incubated for 18-24 hours at 37'C under normal atmosphere, CO-,-enriched atmosphere in GasPak jars, or anaerobically in GasPak jars.

EXAMPLE 1 Acidification of saliva in vitro by the action of the native microbial flora on sugars

Figure IA shows the initial pH of successive rinses of the mouth of a human subject: the first with water; the next nine with 5% glucose solution. Lastly, six samples of undiluted saliva were collected. The initial pH of the samples (measured immediately after collection) fell from 6.8 to about 6.4 with successive glucose rinses. Upon termination of rinsing with glucose solution, the pH of the saliva returned to, and somewhat overshot, the starting value prior to exposure to sugar solutions.

The first water rinse and the nine glucose rinses were incubated for almost 6 h and the pH was measured at the indicated intervals. The results shown in Figure IB illustrate that the pH of the water rinse remained at or slightly above the initial value of 6.8, whereas the pH of the various glucose rinses remained steady for approximately 2 h and then dropped by about 2 units over the following 4 h. The rate of acidification was quite similar among the various glucose-rinse samples. As shown in figures IC and ID, a pH drop was also observed when saliva was incubated in vitro with 5% sucrose or 5% fructose.

These results show that saliva containing its native bacterial population becomes acid upon incubation with glucose or other fermentable sugars in vitro, indicating that this experimental system is useful as a model to study the effects of antimicrobial agents on the fermentation of sugar by oral bacteria.

EXAMPLE 2

Effect of nisin on sugar fermentation in saliva

Saliva was collected after a series of rinses of the mouth of a human subject with a 5% glucose solution. The rinses were combined to form a homogeneous pool , which was then divided into aliquots to which different amounts of nisin were added. The results are shown in Figure 2A and demonstrate that nisin concentrations between 1 and 5 μg/ml significantly reduced the rate and the extent of acidification, as compared to the control containing no nisin, whereas nisin concentrations of 10 and 30 μg/ml totally suppressed the drop in pH over a 6 h observation period.

Similar experiments were performed using sucrose and fructose instead of glucose. The results shown in Figures 2B and 2C demonstrate that nisin retarded or suppressed the acidification of saliva under these conditions as measured by a change in pH.

These results demonstrate that low concentrations of nisin inhibit fermentation of sugars by salivary bacteria.

EXAMPLE 3 Effect of nisin on viability of native salivary bacteria Using the same experimental design as in Example 2 , saliva was pooled from glucose rinses. Aliquots of the rinses were plated onto agar plates and the viability of total salivary bacteria was determined after incubation of plates under aerobic and anaerobic conditions and in a 5% C0₂-enriched atmosphere. These different techniques were used so as to detect the various populations of bacteria present in the saliva.

As shown in Table 1, even at the highest concentration of nisin tested (30 μg/ml) , there were only moderate drops in apparent viability at early time points and the cells tended to recover at later times. The pH was measured in parallel; as shown in Figure 3, the effect of different concentrations of nisin in suppressing glucose fermentation was similar to what was observed in Example 2.

These experiments show that concentrations of nisin sufficient to suppress or retard acid formation in saliva containing sugars do not significantly affect the viability of oral bacteria present in saliva.

EXAMPLE 4

The mouth of a human subject was repeatedly rinsed with 5% glucose solution containing 10 μg/ml of nisin. The final concentration of nisin in the expectorated rinse was less than 10 μg/ml and estimated at 3 to 7 μg/ml, due to dilution of the rinse solution with the saliva. The rate of acidification was monitored over time by measuring the pH of each individual rinse. As shown in Figure 4, suppression or delay of acidification was observed. The effect was more evident beginning with the second glucose rinse. The results in Figure 4 show that when nisin is introduced directly into the mouth in a sugar solution, it exerts a similar suppressive effect on acidification of saliva to that previously observed when it was added to saliva in vitro. From a comparison of the first and later rinses with glucose solution containing nisin, it would appear that inhibition of acidogenesis is potentiated upon repeated exposure to nisin. Repeated exposure would in fact occur during the act of drinking a beverage or chewing a food containing nisin. Table 1. Effect of different concentrations of nisin on the viability of salivary bacteria

CFU/ml

Nisin Time Aerobic CO, Anaerobic

(μg/ml) (min) 0 0 2.8X10⁷ 7.3X10⁷ 4.5X10⁷

5 4.0X10⁷ 6.4X10⁷ 3.1X10⁷

30 4.0X10⁷ 1.3X10⁸

165 5.6X10⁷ 6.9X10⁷ 300 1.4X10⁸ 7.9X10⁷ 5.5X10⁷

3 5 2.7X10⁷ 1.8X10⁷ 3.6X10⁷

30 2.0X10⁷ 2.2X10⁷ 2.9X10⁷

165 3.8X10⁷ 8.0X10⁷ 1.1X10⁸

300 1.6X10⁸ 1.8X10⁸ 3.0X10⁸

5 5 2.6X10⁷ 3.0X10⁷ 2.0X10⁷

30 2.3X10⁷ 1.3X10⁷ 2.0X10⁷

165 1.5X10⁷ 4.5X10⁷ 5.2X10⁷

300 1.4X10⁸ 8.2X10⁷ 1.8X10⁸

10 5 9.8X10⁶ 1.2X10⁷ 1.8X10⁷

30 2.1X10⁷ 1.0X10⁷ 1.6X10⁷

165 2.8X10⁷ 2.1X10⁷ 4.0X10⁷

300 1.6X10⁸ 8.2X10⁷ 1.5X10⁸

20 5 9.0X10⁶ 1.9X10⁷ 2.0X10⁷

30 8.5X10⁶ 6.6X10⁶ 3.1X10⁶

165 1.3X10⁷ 1.2X10⁷ 1.2X10⁷

300 >10⁸ >10⁸ >10⁸

30 5 7.7X10⁶ 1.5X10⁷ 1.3X10⁷

30 5.9X10⁶ 3.8X10⁶ 1.6x10°

165 2.2X10⁷ 8.9X10° 1.7X10⁶

300 6.8X10⁷ 4.1X10⁷ 3.2X10⁷ EXAMPLE 5

A specific embodiment of the invention is the incorporation of nisin into fruit-flavored candies and beverages containing citrate in order to deliver a sub- bactericidal concentration into the oral cavity. Because citrate (as well as other chelating agents) potentiates the activity of nisin against some microorganisms, the effect of nisin was measured in the presence of 3 mM sodium citrate (Na₃C₆H₅0-«2H₂0) on the fermentation of glucose by salivary bacteria. A pool of saliva from 5% glucose washes of the mouth of a human subject was divided in two and one portion was made 3 mM in citrate. Both portions were further divided for exposure to different nisin concentrations.

Comparing Figure 5A (no citrate) and Figure 5B (citrate added) , it is clear that, in the absence of nisin, the pH drop was slower in the presence of citrate. This may be due to the buffering capacity of citrate (i.e., there is a more extensive fermentation needed to produce a drop in pH) and/or to a slight effect of citrate itself on bacterial metabolism. In any event, low concentrations of nisin (1 and 3 μg/ml) were more effective in the presence of citrate than in its absence in retarding acidification of the saliva.

These experiments demonstrate that citrate does not interfere with the action of nisin in inhibiting sugar fermentation by bacteria and, in fact, appears to enhance this activity. Therefore, it is possible that the amount of nisin that must be added to sweet foods or beverages, in order to produce the desired suppression of acid formation by oral bacteria, may be lower for foods or beverages containing citrate or other chelating agents such as (but not limited to) EDTA, succinate and hydroxybenzoate, whether the chelating agent is added exogenously or present naturally (for example, in the case of citrate, in fruit-based foods) .

Claims

We claim:

1. A method for controlling the fermentation of sugars by bacteria present in the oral cavity of a mammal, which comprises administering to the oral cavity a composition comprising a lanthionine-containing bacteriocin in an amount which is not sufficient to compromise the viability of the bacteria but which is adequate to inhibit the metabolism of the bacteria so as to suppress the fermentation of sugars.

2. A method for preventing or reducing the promotion of tooth decay by sugars in a mammal, which comprises administering to the oral cavity of the mammal a composition comprising a lanthionine-containing bacteriocin in an amount which is not sufficient to compromise the viability of the bacteria but which is adequate to inhibit the metabolism of the bacteria so as to suppress the fermentation of sugars.

3. A method according to claim 1 or 2 wherein the lanthionine-containing bacteriocin is incorporated into a sugar- or complex-carbohydrate-based food, confectionery or beverage.

4. A method according to claim 1 or 2 wherein the lanthionine-containing bacteriocin is applied as a surface coating or dusting to the surface of a sugar- or complex- carbohydrate-based food or confectionery.

5. A method according to claim 1 or 2 wherein the lanthionine-containing bacteriocin is incorporated into a mouthrinse, spray, gum or lozenge designed for use following consumption of sugar- and complex-carbohydrate-containing foods, confectioneries and beverages.

6. The method according to claim 3 wherein the effective concentration of the bacteriocin is in the range from about 0.1 μg/ml to about 100 μg/ml or from about 0.1 ppm to about 100 ppm.

7. The method according to claim 4 wherein the effective concentration of the bacteriocin is in the range from about 0.1 ppm to about 100 ppm.

8. The method according to claim 5 wherein the effective concentration of the bacteriocin is in the range from about 0.1 μg/ml to about 100 μg/ml or from about 0.1 ppm to about 100 ppm.

9. A sugar- or complex-carbohydrate-based food, confectionery or beverage further comprising a lanthionine- containing bacteriocin in an effective concentration range from about 0.1 μg/ml to about 100 μg/ml or from about 0.1 ppm to about 100 ppm.

10. A mouthrinse, spray, gum or lozenge further comprising a lanthionine-containing bacteriocin in an effective concentration range from about 0.1 μg/ml to about 100 μg/ml or from about 0.1 ppm to about 100 ppm.