# Patent

## Title
Method of shaping a spherical body

## Abstract

The present invention relates to the discovery that hop extract is useful as an antibacterial agent against the dangerous pathogens Clostridium botulinum, Clostridium difficile, and Helicobacter pylori at levels below that at which a flavor from the acids contained therein is objectionable. More specifically, a process and associated product is described herein, comprising applying a solution of hop extract to a food, beverage or other medium so that the final concentration of hop ingredients is about 1 ppm or higher in order to inhibit the growth of Clostridium botulinum, Clostridium difficile, and/or Helicobacter pylori.

## Background
The present invention relates to the use of hop extracts for controlling Clostridium botulinum, Clostridium difficile, and Helicobacter pylori.

The most prevalent groups of bitter acids found as components of hops are the alpha-acids and beta-acids, also referred to as humulones and lupulones, respectively. Both contribute bitterness to beer, but the alpha-acids are much more intense in this regard than the beta-acids. Producers of hop extracts have recently used liquid carbon dioxide under supercritical conditions. A by-product of the operation is a product which contains approximately 61 weight percent beta-acids, the remainder consisting essentially of other hop resins.

Quite apart from their use in beer, hops and hop acids have also been recognized as microbial inhibitors. More specifically, hop acids and resins have been shown to be primarily active against some gram positive bacteria and Mycobacteria. Activity against gram negative bacteria is far less pronounced. It has been suggested that the reduced effect was due to induced permeability of the cell membrane in gram positive bacteria, but was inactivated by the serum phosphatides in gram negative bacteria. Arch. Mikrobiol. 94, pp. 159-171, 1973.

Other more recent references have been identified, such as U.S. Pat. No. 5,286,506 (1994) and Larson, Yu, Price, Haas and Johnson, International Journal of Food Microbiology, 1996, which report on the use of beta acids as extracted from hops for controlling Listeria. More specifically, 6 to 50 ppm of beta acids, as extracted from hops, was found in media to protect against Listeria monocytogenes contamination, while in foods, depending on the specific food, higher levels (100-300 ppm) were necessary.

Attention is also directed to the following references: Agricultural and Biological Chemistry, Vol. 49, No. 2, pp 399-404 (1985) which discloses that humulone, lupulone and related compounds were found to have antifungal activities; Dissertation Abstracts, Vol. 53-08B, 1991, pp. 3861, reports that compounds derived from hops possess antibacterial activity, and more specifically, the antibacterial activity against Lactobacillus brevis was found to be pH-dependent; Journal of the Institute of Brewing, 99 (5) 405-411 (1993) reports on the results of studies investigating the ability of hop acids to inhibit beer spoilage activity; Journal of the Institute of Brewing, 99 (1) 43-48 (1993) reports on the antibacterial activity of hop bitter resins derived from recovered hopped worts. More specifically, strains of thermophilic Bacillus spp were identified which failed to grow in certain sweet worts derived from mashes to which centrate (recovered hop wort) had been added; J. Food Prot. Vol 57, No. 1, pp 59-61 (1994) reports on the antimicrobial activity of hop resins against Streptococcus salivarius. The two hop resins used were iso-alpha acid and beta resin; Agric. Biol. Chem., Vol. 49, No. 2, pp. 399-403 (1985) discloses that humulone, lupulone and related compounds as having antifungal activities; Lebensm. Ind. Vol. 28, No. 7, pp. 311-315 (1981) reports that tests showed that hop extract and isomerized hop extract have similar anti-microbial properties like hops, but the antimicrobial effect of the hops in beer production was low. J. Appl. Bacteriol., Vol. 72, No. 4, pp. 327-324 (1992) considered the antibacterial effect of weak acids derived from the hop plant Humulus lupulus. The antibacterial activity of trans-isohumulone was about 20 times greater than that of humulone, 11 times greater than colupulone, and nine times greater than that of trans-humulinic acid when the degree of ionization was taken into account.

However not all gram positive bacteria are sensitive to hop resins as is well known to the Brewer and see J. Fernandez and Will Simpson in J. App Bacteriology, 75 315-319 (1993). Also G. Haas and B. Barsoumian in J. Food Protection 57, 59-61 (1994) worked with a strain of Bacillus subtilis which was resistant.

None of the art noted above deals with the control of botulism, which is well-known as an acute intoxication manifested by neuromuscular disturbances after ingesting food containing a toxin elaborated by Clostridium botulinum. The causative agent is actually one of several types of exotoxins elaborated by the sporulating, anaerobic bacillus Clostridium botulinum, which causes human poisoning. Botulinum toxins are highly poisonous proteins resistant to digestion by gastrointestinal enzymes. Clostridium difficile is one of the major causes of diarrheal disease particularly in elderly humans treated with antibiotics. Very few antibiotics are effective and treatment of this infection is difficult at best. Only vancomycin of the well known antibiotics seems to be useful in treatment. Helicobacter pylori is a common cause of gastric ulcers and chronic active gastritis in humans. Ulcer relapses are common in humans treated with antibiotics or bismith nitrate. Other intervention strategies have to be sought and a nutritional or dietetic approach would be highly desirable.

The present invention relates to the discovery that hops extract or the components of hops extract are useful as an antibacterial agent against dangerous pathogens Clostridium botulinum, Clostridium difficile, and Helicobacter pylori. More specifically, a process and associated product is described herein, comprising applying at least about 1 ppm or greater, by weight, of beta acids, or hop extracts to inhibit growth of Clostridium botulinum, Clostridium difficile, and Helicobacter pylori. Medications, disinfectant solutions or pharmaceutical compositions containing these materials may also be used.

FIGS. 1A, 1B and 1C illustrate the inhibition of Clostridium botulinum 56A by hop extracts "a" (41% beta, 12% alpha and 47% desoxy alpha, hop oils and hop waxes), "b" (65% w/v beta acids) and "c" (6% w/v post beta-acids in Tween 80), at different concentrations in ethanol (50%) solution.

FIGS. 2A, 2B and 2C illustrate the inhibition of Clostridium botulinum 62A by hop extracts a, b and c, as described above.

FIGS. 3A, 3B and 3C illustrate the inhibition of Clostridium botulinum 213B by hop extracts a, b and c, as described above.

FIGS. 4A, 4B and 4C illustrate the inhibition of Clostridium botulinum Lamanna-Okra B by hop extracts a, b and c, as described above.

FIGS. 5A, 5B and 5C illustrate the inhibition of Clostridium botulinum Alaskan E by hop extracts a, b and c, as described above.

FIGS. 6A, 6B and 6C illustrate the inhibition of Clostridium botulinum Beluga E by hop extracts a, b and c, as described above.

FIGS. 7A, 7B and 7C illustrate the inhibition of Clostridium botulinum 17 by hop extracts a, b and c, as described above.

FIGS. 8A, 8B and 8C illustrate the inhibition of Clostridium botulinum 4848B by hop extracts a, b and c, as described above.

FIGS. 9A, 9B and 9C illustrate the inhibition of Clostridium difficile 43255 by hop extracts a, b and c, as described above.

FIGS. 10A, 10B and 10C illustrate the inhibition of Clostridium difficile 10463 by hop extracts a, b and c, as described above.

The present invention relates to the discovery that hop extracts or fractions are useful as a preservative inhibiting the pathogens Clostridium botulinum, Clostridium difficile, and Helicobacter pylori and as agents to prevent illness caused by said pathogens. Three different hop extracts were evaluated to demonstrate the broad applicability of the present invention.

The hop extracts as used herein may comprise solvent extracted hops, or liquid CO.sub.2 or supercritical CO.sub.2 gas extracted hops. Particularly preferred are CO.sub.2 liquid or CO.sub.2 critical gas extracts. Generally, the hop extracts are added to a food product or other vehicle, in solution, to achieve at least about one part per million, by weight, of beta acids in the GI tract or stomach. Amounts less than about 1 ppm, by weight, beta acids, does not appear to provide protection against Clostridium botulinum and Clostridium difficile. The solution preferably contains about 5 ppm-100 ppm, by weight, of beta acids. The upper level is dictated by taste and solubility.

FIGS. 1-10 collectively illustrate the experimental results confirming the antimicrobial effects disclosed herein. More specifically, and as noted above in the brief description of the drawings, FIG. 1A through 10A reference the use of hop extract "a", which contained 41% beta, 12% alpha and the remaining 47% contained a mixture of desoxy-alpha, hop oils and hop waxes. FIGS. 1B-10B refers to the use of hop extract "b", which contained 65% (w/v) beta acids. FIG. 1C-10C refer to hop extract "c" which contained 6% (w/v) post beta acids in Tween 80. In each case the hop extract was made up as a solution in 50% ethanol, and added to achieve 1, 5, 10, 50 and 100 ppm. A control with 50% ethanol but without hop resin was included.

The organisms targeted in this invention included 8 strains of Clostridium botulinum and two strains of Clostridium difficile, as listed below: Clostridium botulinum: Proteolytic type A:        56A, 62A Proteolytic type B:        213B, Lamanna-Okra B, Non-proteolytic type B:    17B, 4848B, Non-proteolytic type E:    Alaska E, Beluga E Clostridium difficile: 43255 10463

These organisms are toxicogenic and have been involved in human intoxication or infections. The inhibition of Clostridium botulinum by hop extracts in broth media was established as follows:

Eight strains of Clostridium botulinum were inoculated as spores separately into tubes of 10 ml trypticase peptone-glucose-yeast extract (TPGY) broth containing 5 different levels (1, 5, 10, 50 and 100 ppm) of three hop extracts. Before inoculation, spores were treated with a heat treatment to activate them in order to achieve maxiumum germination. For proteolytic strains, spores were heat treated at 80.degree. C. for 10 min and spores from non-proteolytic strains were treated at 60.degree. C. for 20 min. Dilutions were made to have an initial inoculum ranging between 2.times.10.sup.3 and 3.times.10.sup.3 spores/ml.

Clostridium difficile strains were incubated in Brain Heart Infusion (BHI), 0. 1% Yeast Extract (UYE) broth at 37.degree. C.

As noted, hop extracts "a", "b" and "c" were tested at five different concentrations in the final medium: 1, 5, 10, 50, and 100 ppm. The tubes were incubated at 30.degree. C. for one week. Growth (measured as increased absorbance) was monitored by optical density (O.D. at 660 nm) at one, two and seven days. Controls (only broth) and ethanol controls were inoculated with the spores but hop extracts were not added. All combinations of variables were tested in duplicate and replicated at least once.

With attention now directed at FIGS. 1A, 1B and 1C through 10A, 10B and 10C, as illustrated therein, hop extracts "a" and "b" produced inhibitory activity towards all eight Clostridium botulinum strains at a concentration as low as 1 ppm, and more preferably at concentrations of 5, 10, 50 and 100 ppm. Accordingly, 5-100 ppm of hop extracts "a" and "b" were found as the most preferred in the broad context of the present invention as applied to the Clostridium botulinum strain. Similarly, spores of Clostridium difficile strains were inhibited by hop extracts "a", "b" and "c" also at concentrations as low as 1 ppm, and more preferably at concentrations of 5, 10, 50 and 100 ppm, establishing effectiveness at the similar preferred range of 5-100 ppm.

The results above confirm that with regards to botulinum, hop extracts, quite apart from the known use in beer, have proven to be uniquely suited to provide effective inhibitory activity against this very important food pathogen. In addition, hop extracts also have shown their inhibitory activity against Clostridium difficile strains. The hop extracts therefore may be conveniently incorporated into a food product by dipping or spraying the food product with a solution of the extracts or alternatively added to a suitable vehicle such as an oral formulation to treat or prevent disease caused by the above microbes.

The following experimental procedure was applied with respect to confirmation of the inhibition of growth of Helicobacter pylori by hop extracts in broth media: Hop extracts "a" and "b" were dissolved in 95% EtOH, filter sterilized through a 0.45 .mu.m syringe filter, and further diluted in filter sterilized 95% EtOH. Ten ml tubes of trypticase soy broth were prepared by adding 0.1 ml of the appropriate dilution of hop extract per 10 ml tube to obtain final concentrations of 1, 5, 10 or 100 ppm hop extract. Controls were prepared by adding 0.1 ml of filter sterilized dH.sub.2 O per 10 ml tube. Ethanol controls were also prepared by adding 0.1 ml filter sterilized 95% EtOH per tube.

An 18 hour overnight culture of Helicobacter pylori (ATCC 43504) in tryptic soy broth (TSB) was inoculated (0.1 ml per 10 ml TSB) into prepared TSB tubes. Caps were loosened on tubes, which were incubated at 37.degree. C. in anaerobe jars containing BBL CampyPak Plus packets, which created a microaerophilic system in the jars. Growth was checked by monitoring optical density at 660nm every day for 3 days. Initial inoculum level (3.8.times.10.sup.5 CFU/ml) was determined by diluting inoculum in 67 mM sodium phosphate buffer and pour plating onto Plate Count Agar, which was incubated 24 hours at 37.degree. C. The results are provided below in Table I: RESULTS Day 1        Day 2        Day 3 control (0 ppm)         G            G            G EtOH control          G            G            G 1 ppm HE#2           NG           NG           NG 5 ppm HE#2           NG           NG           NG 10 ppm HE#2          NG           NG           NG 100 ppm HE#2         NG           NG           NG 1 ppm HE#3            G            G            G 5 ppm HE#3           NG           NG           NG 10 ppm HE#3          NG           NG           NG 100 ppm HE#3         NG           NG           NG G = growth NG = no growth

Growth of H. pylori was completely inhibited in TSG at 37.degree. C. over 3 days by hop extract #2 at levels as low as 1 ppm, and by hop extract #3 levels as low as 5 ppm.

As can be seen from the above, growth of Helicobacter pylori was completely inhibited in TSB at 37.degree. C. over 3 days by hop extract "a" at levels as low as 1 ppm, and by hop extract "b" at levels as low as 5 ppm.

In addition to the above, those skilled in the art will recognize herein that the present invention also relates to the preparation of disinfectant compositions to inhibit growth, and pharmaceutical compositions to prevent transmission, of the pathogens identified herein, wherein said compositions comprise at least I ppm of hop extracts, or more preferably, 5, 10, 50 and 100 ppm, and/or the specific range between about 5-100 ppm.

## Claims

1. A method for inhibiting the growth of Clostridium dfficile and/or Clostridiuxm botulinum in a food or beverage product which contains Clostridium difficile and/or Clostridium botulinum wherein the method comprises adding a hop extract which is a solvent or CO.sub.2 extract containing at least 1 ppm by weight beta-acids to the food or beverage product whereby the growth of Clostridium difficile and/or Clostridium botulinum is inhibited.

2. The method of claim 1, wherein hop extract is added as an ethanolic solution.

3. The method of claim 1, wherein said hop extract contains 5-100 ppm by weight.

4. method of claim 1, wherein said hop extract comprises a mixture of (1) beta-acids, and (2) a hop extract material selected from the group consisting of an alpha-acid, a desoxy-alpha acid, a hop oil, a hop wax and a mixture thereof.

5. The method of claim 4, wherein said mixture comprises 65 wt. % beta-acids.

6. The method of claim 1, and including the step of adding a surface active agent to a solution in order to solubilize said hop extract prior to adding said hop extract to said food or beverage product.