Field of the Invention:

The present invention is directed to a composition containing a compound produced by the reaction of glycine or a salt of glycine with formaldehyde which effectively inhibits microbial growth in a variety of substances. Description of the Prior Art:

Reports on the reaction of glycine with formal¬ dehyde is limited to work done over 50 years ago. W. Lob [Biochem. Z., 51, pp. 116-127 (1913)] reported that glycine

(also called aminoacetic acid and glycocoll) plus formaldehyde formed the Schiff base in neutral solution and formed methylenediglycine, isolated, as the dihydrochloride, in acid solution. Lob did not report isolating a product from reaction in alkaline solution. Following L ^' όb, H. Krause

[Ber. , 52_, pp. 1211-1222 (1919)] described the preparation of sodium or potassium salts of hydroxymethylglycine by low temperature reaction in aqueous solution. Krause also described the preparation of other salts, e.g., barium, lead, silver, copper, mercury and calcium salts, of hydrox meth l- glycine from either glycine salt plus formaldehyde or by replacement of the sodium or potassium cation of the hydroxy- ^' _. methylglycine salt.

More recently, N-hydroxymethylglycine is disclosed in U.S., Patent 3,291,606. In that patent, a photographic color image is reported to be protected against fading and discoloration caused by intense tungsten illumination when bathed in an aqueous solution of glycine, glycylglycine or N-hydroxymethylglycine. The source of the N-hydroxymethyl- glycine is not disclosed.

Recent survey articles discussing the reaction between glycine and formaldehyde have stressed the complexity of the chemistry. See, e.g., Rodd, The Chemistry of Carbon Compounds, 2nd Ed. Vol ID (1965) p. 167. For example, while the condensation of the sodium salt of glycine with

formaldehyde may be viewed as a simple addition reaction:

COO~Na ⁺ C00 ^" Na ⁺

I +CH 0 I

CH ₂ H ₂ * CH ₂ -NH-CH ₂ 0H

it can also be much more complicated because the hydroxy¬ methyl derivative could dehydrate to a Schiff base, react with another molecule of glycine Or polymerize. Thus, rather than term the reaction product simply sodium hydroxy methylaminόacetate, it is more accurate to speak of it as an equilibrium mixture of compounds produced by the reactio of the sodium salt of glycine with formaldehyde. The precise proportions of the particular compounds in the mixture will depend on the particular reaction conditions. While it is evident that the prior art has long recognized that salts of glycine could be reacted with formaldehyde, there is little disclosure regarding the utility of the reaction product.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the inven¬ tion to utilize the product formed by the reaction of glyci or a salt of glycine with formaldehyde in a new and unobvio manner as a biocidal agent which inhibits the growth of micro-organisms in substances requiring icrobial inhibitio

Another object of the invention is to provide a ^* new and improved biocidal composition containing the reacti product as the primary active ingredient against microbial growth.

Other objects and advantages of the invention wil be evident to those of skill in the art upon review of the entire disclosure contained herein.

These objects and advantages -are accomplished by providing a biocidal composition consisting essentially of the product produced by the reaction of glycine or a salt of-glycine with formaldehyde. By incorporating an effectiv amount of this composition into a substance requiring

CϊvIP

^■

microbial inhibition, the substance can be protected against contamination by a variety of microorganisms including bacteria, yeast and mold.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that the product produced by the reaction of glycine or a salt of glycine with formaldehyde is a highly effective biocidal agent which inhibits microbial growth in a variety of applications whether they be in solid, liquid or emulsion form. Such applications include, but are not limited to, cosmetics, foodstuffs, pharmaceuticals, paints, cutting oils or fluids, agricultural products, oil drilling fluids, paper industry, embalming solutions, cold sterilization medical and dental equipment, cooling towers, fabric impregnation, latexes, swimming pools, inks, household disinfectants, waxes and polishes, toilet bowl cleaners, bathroom cleaners, laundry detergents, soaps, wood preservatives, hospital and medical antiseptics and adhesives.

In addition to the glycine-formaldehyde reaction product, the biocidal composition of the invention may contain other conventional ingredients including other microbial agents, suspending agents, wetting agents, anti- scaling agents, corrosion inhibitors and pH control agents. The resulting biocidal composition is incorporated into the substance for which protection is desired in an amount which is effective for inhibiting microbial growth. Of course the amount of the composition incorporated into the substance as well as the particular additional ingredients will vary depending upon the substance being protected and the environment of use. Generally, substances containing less than 1.0% by weight will be effectively protected from microbial growth in most environments. Principally for economic reasons, concentrations less than about 0.5% are preferred. Those of skill in the art will have little difficulty in selecting specific amounts required for

particular uses based upon the information contained herein, especially in the examples.

In preparing the biocidal composition of the invention, a mixture of glycine and a basic reagent, such as an alkali metal hydroxide, is prepared and mixed with formaldehyde. To obtain a salt other than the alkali metal salt, a compound which will cause replacement of the alkali metal cation is added to the reaction mixture. In this manner numerous salts can be prepared including aluminum hydroxymeth laminoacetate, calcium hydroxymethylaminoacetat magnesium hydroxymethylaminoacetate, copper hydroxymethyl¬ aminoacetate and zinc hydroxymethylaminoacetate. When alk substituted glycinates are reacted in place of glycine, alkyl hydroxymeth laminoacetates are produced. After the reaction is complete, an equilibrium mixture of .reaction products is obtained as a relatively clear, aqueous solution. The liquid product may be dried to produce a powder, crystalline composition. The powder is the preferred form of the composition of the invention since it is easier to handle than a liquid. When the situa tion requires a liquid formulation, the powder can be readily dissolved in aqueous media.

The composition of the invention thus provides a highly effective means for preserving substances susceptibl to microbial contamination by significantly inhibiting growth of bacteria, yeast and mold. It is readily incor¬ porated into a variety of substances having a variety of forms including liquids, solids and emulsions.

To further illustrate the various aspects of the invention, the following examples are provided. However, it is to be understood that their purpose is entirely illustrative and in no way intended to limit the scope of the invention. In the examples all percentages are ^* given b weight unless otherwise noted.

EXAMPLE 1 Preparation of Sodium Hydroxymethylaminoacetate

A solution of 75 g. (1.00 mole) of glycine in 100 g. (1.00 mole) of 40% aqueous sodium hydroxide was treated with 82 g. (1.00 mole) of 37% formaldehyde holding the batch temperature below 50°C by water cooling. The final solution pH was 11.4. Water was removed slowly at . room temperature, and the crystals which formed were col¬ lected, washed with cold water, and dried, giving 47 g. (37% yield) of white solid, mp 150-155"dec. Anal. Found: C, 26.54; H, 4.81; N, 10.86.

EXAMPLE 2 Preparation of Potassium Hydroxymethylaminoacetate

To a solution of 84 g. {1.5 moles) of potassium hydroxide in 127 g. of water was added 112.5 g. (1.5 moles) of glycine. With stirring and cooling, 121.8 g. (1.5 moles) of 37% formaldehyde solution was added slowly at 30-35°C. The clear colorless solution, pH 11.4,- was dried by removal of water, leaving a hygroscopic white powder, mp 162-184°C. dec. Anal. Found: C, 24.35; H, 3.97; N, 10.13.

EXAMPLE 3 Preparation of Aluminum Hydroxymethylaminoacetate

To a stirred and water-cooled mixture of 90 g. .(1.2 moles) of glycine in 96 g. (1.2 moles) of 50% aqueous sodium hydroxide was added 98.4 g. (1.22 moles) of 37% formaldehyde over 30 minutes. The batch temperature was allowed to rise to 60°C. After 20 minutes stirring, the batch was filtered and the clear, colorless solution was allowed to stand at room temperature overnight. A solution of 96.6 g. (0.4 mole) of aluminum chloride hexahydrate in 150 ml. of water was added with stirring, and the precipi¬ tate was collected and washed. The product was air-dried overnight to give 172 g. of aluminum hydroxymethylaminoacetate.

EXAMPLE 4 Methyl Hydroxymethylaminoacetate

A solution of 6.3 g. (0.05 mole) methylglycinate hydrochloride in 4.0 g. (0.05 mole) of 50% aqueous sodium

hydroxide plus 10 ml. of water was treated with 4.1 g.

(0.05 mole) of 37% formaldehyde. The warm solution was stirred at room temperature for 30 minutes to give a solution, pH 6, of methyl hydroxymethylaminoacetate. EXAMPLE 5 ϋs.e as an Antimicrobial Preservative: Challenge Testing a 0.3% Solution of Sodium Hydroxymethylaminoacetate With

Various Microorganisms

The following procedure for measuring critical killing time was carried out using a gram-positive bacteriu (S. aureus ATCC 6538) , two gram-negative bacteria (P. aerug nosa ATCC 15442 and E. coli ATCC 10536) , a yeast (C. albica ATCC 10231), and a mold (A. niger ATCC 9642)-. Bacteria A 24 hour A.O.A.C. broth culture was used for the test. 0.5. ml. of the 24 hour culture was added to 4.5 ml. of the test sample and mixed thoroughly. • The sample was th stored at 35°C in a hot air incubator for the duration of t test. At pre-selected time intervals of 24, 48 and 72 hour a loopful (0.1 ml.) of the sample was aseptically removed f the incubator and placed in A.O.A.C. broth with Letheen. T tubes containing the inoculated broth were incubated for 48 hours at 35°C and then examined for microbial growth.

If the sample turned the medium cloudy on the initial subculture, the subculture was incubated for 24 hou at 35°C and thereafter subcultured again into fresh medium and incubated for 48 hours at 35°C. Yeast

The same procedure was used as for bacteria, except Sabourand Liquid Medium with Letheen was used in place of A.O.A.C. broth with Letheen. Molds

The growth of a 7-10 day slant was washed off wit 10 ml. of sterile saline. 0.5 ml. of this suspension was added to 4.5 ml. of the sample and mixed thoroughly. The sample was stored at room temperature for the duration of the' test. At pre-selected time intervals, a loopful (0.1 m was removed and placed into Sabourand Liquid Media with

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Letheen and incubated for 7 days at room temperature and then examined for microbial growth.

If the sample turned the medium cloudy on the initial subculture, the subculture was incubated for 24 hours at room temperature. After the 24 hour incubation, the test tube was subcultured again into fresh medium and incubated for 7 days at room temperature.

The results of challenge testing a 0.3% aqueous solution of sodium hydroxymethylaminoacetate were as follows:

Subculture After Incubation. Times (Days) *

Species ATCC Number JL ^ 3

P. aeruginosa 15442 0 0 0 E. coli 10536 0 0 0 S. aureus 6538 0 0 0 C. albicans 10231 0 0 0 A. niger 9642 0 0 0

*Code: + = ^** Growth, 0 = No Growth

EXAMPLE 6

Use as a Preservative for Cosmetics A model shampoo formulation (a water base deter¬ gent system) containing the following ingredients:

Ingredient % by Weight

Triethanolamine lauryl sulfate (40%) 25.00

Lauryl Diethanolamide 5.00

Amphoteric-2 5.00

Polyoxyethylene Lanolin (50%) 3.00

Phosphoric Acid 0.20

Demineralized Water qs to 100% was challenge tested without added preservative and also with added 0.2% sodium hydroxymethylaminoacetate, following the testing procedure described in Example 5. The shampoo was found to be subject to microbial contamination in the absence of preservative, but well-preserved when containing 0.2 sodium hydroxymethylaminoacetate.

— 77ΪL_ ^* V.'if

A. Shampoo formulation without added preservative

Subculture After Incubation Time (Days) * Species ATCC Number 1 2 3 7

P. aeruginosa 15442 + + + +

E. coli 10536 + + + +

S. aureus 6538 + + 0 0

C. albicans 10231 + + + 0 A. niger 9642 + + + +

*Code: + = Growth, 0 = No Growth

B. Shampoo formulation containing 0.2% sodium hydroxy¬ methylaminoacetate

Subculture After Incubation Time (Days) *

Species ATCC Number 1 2 3 7

P. aeruginosa 15442 0 0 0

E. coli 10536 0 0 0

S. aureus 6538 0 0 0

C. albicans 10231 0 0 0

A. niger 9642 + 0 0

*Code: + = Growth, 0 = No Growth EXAMPLE 7

Use as a preservative for cosmetics A commercial cosmetic emulsion which .frequently b contaminated with various "house" microorganisms during manufacture was reformulated to contain 0.3% sodium hydroxymethylaminoacetate. Standard challenge testing of 0.3% aqueous solutions of sodium hydroxymethylamino¬ acetate and of the.cosmetic emulsion itself containing 0.3% sodium hydroxymethylaminoacetate with these problem "house" microorganisms showed complete kill within 3 days. In fact, in the cosmetic emulsion itself, the two "house" microorganisms which were the most difficult to kill were killed within 7 days by sodium hydroxymethylaminoacetate at a concentration of only 0.15%.

A. Aqueous solutions of 0.3% sodium hydroxymethyl¬ aminoacetate

Subculture After Incubation Time (Days) *

Species 1 2 3

P. aeruginosa 0 0 0

P. aeruginosa JIT 0 0 0

P. aeruginosa tr-5 + + 0

Yeast + + 0

Mold 0 0 0 ^t Code; + = Growth, 0 = No Growth

B. Cosmetic emulsion containing 0.15% sodium hydroxy- . methylaminoacetate

Subculture After Incubation Time (Days) *

Species 1 2 3 7

P. aeruginosa #3 + + + 0 Yeast - 0 0 0

*Code: + — Growth, 0 = No Growth

EXAMPLE 8

Use as a preservative for adhesives A poster paste ashesive based on a potato starch formulation was found to develop odor and mold when ;stored before use during spring and summer months. Chal¬ lenge testing of the poster paste slurry with the five microorganisms listed in the United States Pharmacoepia XIX, page 588 (1975) for challenging multiple-dose parenteral, otic, nasal and ophthalmic products, showed significant differences between unpreserved poster paste slurry and poster paste slurry containing Q.3% sodium hydroxymethylaminoacetate.

-10- A. Unpreserved poster paste slurry

Subculture After Incubation Times (Days) *

Species ATCC Number 1 2 3 7

P. aeruginosa 9027 + + + +

E. coli 8739 + + + +

S. aureus 6538 + + + +

C. albicans 10231 + + + +

A. niger 16404 + + + + *Code: + = 't , 0 — NO Growth

B. Poster paste slurry containing 0.3% sodium hydroxy¬ methylaminoacetate

Subculture After Incubation Times (Days) *

Species ATCC Number 1 2 3

P. aeruginosa 9027 o - 0 0 E. coli 8739 0 0 0 S. aureus 6538 0 0 0 C. albicans 10231 0 0 0 A. niger 16404 0 0 0

*Code: + = Growth, 0 = No Growth

EXAMPLE 9

Use in lubricating and cutting fluids

A petroleum-based lubricating oil concentrate ,(15 ml.) was diluted to 500 ml. with tap water and inoculated with a bacterial and mold inoculum which had been maintained in a petroleum base cutting fluid and in a synthetic coolant, respectively. The bacterial inoculum consisted mostly of Fusariu and Cephalosporium species. Microbial counts and re-inoculation was done weekly, and all runs were carried out in duplicate. Experiments were carried out at room temperature (25°C) . In the absence of added preservative, the lubricating fluid was found to contain in excess of 10 organisms/ml. at the first weekly subculture assay after inoculation with bacteria and mold. When the diluted lubricant (coolant emulsion) contained 0.1% sodium hydroxymethylaminoacetate, subculture

C.

after three weekly inoculations and re-inoculations showed less than 10 organisms/ml. Only after the fourth weekly inoculation did the coolant emulsion support microbial growth and show microbial levels in excess of 10 organismmss//mmll.. EXAMPLE 10

Antimicrobial effectiveness of hydroxymethylaminoacetic acid and its salts; - preservation of milk.

The microbial spoilage of whole milk at room temperature was prevented by the incorporation of 0.3% hydroxymethylaminoacetic acid or 0.3% hydroxymethylamino¬ acetate salt. The following series of tests was termi¬ nated after 23 days.

Preservative added (at 0.3% ,. . ^iffl ? ^{t0 S} S ^θi ϊ ^ag ; concentration) to whole milk ^(x -? ^« development of odor, color, curdling, etc.)

None 2 days

Hydroxymethylaminoacetic acid 23+ days

Sodium hydroxymethylaminoacetate 23+ days Potassium hydroxymethylaminoacetate 23+ days

Calcium hydroxymethylaminoacetate 23+ days

Copper hydroxymethylaminoacetate 23+ days

Magnesium hydroxymethylaminoacetate 23+ days

Zinc hydroxymethylaminoacetate 23+ days Aluminum hydroxymethylaminoacetate 23+ days

EXAMPLE 11

Antimicrobial effectiveness of hydroxymethylaminoacetic acid and its salts; - preservation of egg

The microbial spoilage of whole egg at room temperature was prevented by the incorporation of 0.3% hydroxymethylaminoacetic acid or 0.3% hydroxymethylamino¬ acetate salt. The following series of tests was terminated after 23 days.

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Time to Spoilage (i.e. development of odor,

Preservative added (at 0.3% and/or color, thickened, concentration) to whole egg separated, etc.

None 2 days

Hydroxymethylaminoacetic acid 23+ days Sodium hydroxymethylaminoacetate 23+ days Potassium hydroxymethylaminoacetate 23+ days

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims.