Liquid cell culture media (nutrient solution) for the detection of viable microorganisms with obligatory product damage potential in beer samples or hop-containing beverage samples.

Abstract

Nutrient solution based on water, preferably demineralized water, produced comprehensively, one or more carbon sources selected from glucose, fructose, sucrose, maltose, dextrins and starch to at least 5 grams / liter; one or more acids selected from phosphoric acid, hydrochloric acid, citric acid, ascorbic acid, sulfuric acid, acetic acid, tartaric acid, malic acid, oxalic acid and lactic acid; one or more protein sources and / or peptide and amino acid sources selected from yeast extract, meat extract, peptone and soy proteins at a minimum of 1 gram / liter; one or more fatty acid sources selected from lecithin, yeast extract, meat extract, glycerin, choline, phospholipids and polysorbate to at least 1 gram / liter; one or more minerals or salts and trace elements selected from sodium chloride, sodium acetate, diammonium phosphate, ammonium sulfate, magnesium sulfate, ammonium citrate, manganese sulfate, dipotassium dihydrogen phosphate and similar substances; one or more inhibitors selected from hop bittering agents (iso-alpha acid), ethanol, carbon dioxide, sulfur and yeast inhibitors (cycloheximide, dehydroacetic acid sodium salt monohydrate); one or more color indicators; and characterized by the fact that the nutrient solution has a pH value of less than 4.8.

Description

[0001] Food and beverage manufacturers must, as part of their quality assurance, check their raw materials, ingredients, intermediate products, and finished beverages for microbiological contamination during production. A fundamental distinction is made between so-called hygiene indicators and germs that are obligatorily harmful to the product.

[0002] Hygiene indicators are microorganisms that can be detected in a sample and thus serve as evidence of their introduction into the process, but do not have a direct impact on the product. No negative changes are expected from these microbes, as they cannot multiply under the product's conditions. Examples include coliform bacteria / E. coli, Legionella, bacilli, and enterococci. These cannot grow in beer, which has an oxygen-free atmosphere, alcohol, acids or an acidic pH (4.3–4.7), and inhibitors such as hop bittering agents (iso-alpha acids), but they can still be present and thus serve as indicators of hygiene deficiencies. These microbes are also referred to as potentially harmful because they do not directly damage the product, but have the potential to disrupt the process or are simply unacceptable.

[0003] Obligately harmful germs are those that are typically capable of multiplying in the finished beverage or its ingredients, thus causing or resulting in alteration or damage to the products. Such germs grow under the conditions present in the sample, even with the use of inhibitors.

[0004] For example, in beer production, quality assurance focuses on so-called beer-spoiling bacteria, which are capable of multiplying under the conditions prevailing in beer. These are germs that thrive in the presence of beer-typical substances such as alcohol, numerous organic acids or a low pH value, hop bittering compounds (iso-alpha acid / iso-beta acid), and CO₂. 2 (without oxygen) they can still grow. These would be, for example, lactic acid bacteria such as Lactobacillus brevis, Lactobacillus casei, Pediococcus damnosus, or also fermenting yeasts as well as over-fermenting foreign yeasts.

[0005] The following method is currently used for detection in the in-house laboratories of breweries and contract laboratories: Liquid enrichment of beer samples. This means that a specific volume of the sample (e.g., 1-200 ml) is placed in a container such as a laboratory bottle and filled with a liquid nutrient solution in the same ratio or greater than the sample volume. These nutrient solutions often have similar compositional properties to the finished beverage, thus enabling the growth of only specific microorganisms through the use of selective factors and inhibitors. The aim of this test is to detect microorganisms that are harmful to the product. This is achieved through microbial growth and subsequent visual evaluation of the increase in turbidity, or microscopic analysis, or DNA analysis such as PCR.

[0006] In this case, the nutrient solutions and even the usual mixtures of nutrient solution and sample are similar, but not sufficiently comparable to the final beverage. The main difference lies precisely in the pH value. pH is an essential value for growth, and because it is also a logarithmic concentration value, it is particularly important.

[0007] For example, if a beer sample (pH value usually between 4.3 - 4.7) is mixed with a nutrient medium (pH value between 5.0-7.9), the pH value is above 4.7 and therefore higher than that of the finished drinks / beers.

[0008] pH values ​​of a selection of internationally used nutrient solutions for the beverage industry: Manufacturer Product name Purpose PH value Doehler NBB-B Beer-spoiling germs 5,5 Doehler NBB-AM Biofilm formers 5,9 Doehler NBB-C Beer-spoiling germs 6,4 Doehler SSL Germs that are harmful to beverages 5,0 Various MRS Lactic acid bacteria 6,2 Doehler Trans-fast Germs that are harmful to beverages 6,1 Difco Micro Inoculum Broth Beer-spoiling germs 6,7 Doemens DS-Select Beer-spoiling germs 5,4

[0009] Beverages from the brewing industry, or hop-containing beverages such as beer, beer-based mixed drinks, non-alcoholic beer, draft soda, etc., typically have a pH value between 4.3 and 4.7 or even lower. This presents a problem: either germs present in the sample that grow at pH values ​​below 4.8 cannot be detected, or germs that would not be viable in the final beverage may grow. In both cases, the analysis must be considered inaccurate.

[0010] In real-world laboratory processes, this regularly leads to deviations, product damage, recalls, and even potential consumer harm. In the author's opinion, no evidence is available on the market that adequately addresses this fact.

[0011] Thus, through extensive research, numerous trials and tests, various formulas and nutrient solutions were developed which, provided certain specifications are met, can solve this problem.

[0012] For analyses of finished hop-containing beverages / beers, the following problem arises: Adding large volumes of a standard nutrient solution to a beverage sample becomes excessively diluted. Therefore, highly concentrated nutrient solutions with a suitable pH value have been developed. These solutions closely resemble the final beverage, requiring only minimal additions to allow the growth of obligate pathogens and thus enable their detection. Simultaneously, the beverage is altered as little as possible, and inhibitory substances are hardly reduced. For example, adding 2 to 5% by volume of concentrated nutrient solution to a beer sample allows for the usual visual detection of obligate pathogens without significantly altering the beer.

[0013] Surprisingly, only germs that are actually or obligately harmful to the product can then grow. False results caused by the growth of "hygiene indicators" or only potentially harmful germs are suppressed as much as possible and can thus be virtually eliminated.

[0014] This also applies to specialty beers with less common characteristics. These include, among others, a high alcohol content and a very low pH value. It also includes beverages containing other inhibitory substances, such as preservatives (e.g., benzoates, sorbates, sulfur, hop bittering agents, oils, aromatics, etc.), which should be altered as little as possible.

[0015] The aim is to simulate the actual, obligatory product damage as accurately as possible, without the influence of hygiene indicators, by precisely adding specific nutrient solutions. Furthermore, processing should be simplified by a high addition of color indicators.

Claims

[1] Nutrient solution based on water, preferably demineralized water, prepared comprising, one or more carbon sources selected from glucose, fructose, sucrose, maltose, dextrins and starch at least 5 grams per liter; one or more acids selected from phosphoric acid, hydrochloric acid, citric acid, ascorbic acid, sulfuric acid, acetic acid, tartaric acid, malic acid, oxalic acid and lactic acid; one or more protein sources and / or peptide and amino acid sources selected from yeast extract, meat extract, peptone and soy proteins at least 1 gram / liter; one or more fatty acid sources selected from lecithin, yeast extract, meat extract, glycerol, choline, phospholipids and polysorbate at least 1 gram / liter; one or more minerals or salts and trace elements selected from sodium chloride, sodium acetate, diammonium phosphate, ammonium sulfate, magnesium sulfate, ammonium citrate, manganese sulfate, dipotassium dihydrogen phosphate and similar; one or more inhibitors selected from hop bittering substances (iso-alpha acid), ethanol, carbon dioxide, sulfur and yeast inhibitors (cycloheximide, dehydroacetic acid sodium salt monohydrate); one or more color indicators; as well as characterized by that the nutrient solution has a pH value of less than 4.

8. [2] Nutrient solution according to claim 1, comprising color indicators which make the growth or proliferation of germs visually visible (color change) and thus simplify the determination and evaluation. [3] Nutrient solution according to one of the preceding claims, characterized by that as the germs grow or multiply, they can be removed from the sample and further analyzed. [4] Nutrient solution according to one of the preceding claims characterized by that they are as filtered as possible or optically clear in order to visually simplify the growth or proliferation of germs through the formation of turbidity or the formation of sediment / precipitates / colonies. [5] Nutrient solution according to one of the preceding claims, characterized by that it has a pH value of less than 4.8, preferably less than 4.

6. [6] Nutrient solution according to one of the preceding claims, characterized by that attention must be paid to the high quantity of ingredients during sterilization already during production, for example autoclaving at only 105-115 °C or using steel sterilization or high pressure disinfection in order to reduce Maillard reactions (discoloration) or turbidity or precipitation or other side reactions. [7] Nutrient solution according to one of the preceding claims, which has a particularly effective formulation, is prepared as follows: a. Carbon source with 10 to 100 grams / liter from fructose, glucose and maltotriose; b. Protein sources with 5 to 50 grams / liter selected from peptones, yeast extract, meat extract; c. Fatty acid sources with 1 to 20 grams / liter selected from yeast extract, meat extract; d. Minerals, salts and trace elements selected from sodium chloride, sodium acetate, magnesium sulfate, ammonium citrate, dipotassium dihydrogen phosphate; e. Undenatured ethanol at 0.5 to 5 vol%; f. Iso-alpha acid from hop extract yielding 10 to 30 BE (bitter units according to EBC / IBU); g. dyes selected from bromocresol green, methyl red, methylene blue; h. Demineralized water; i. Acids to achieve a pH of 4.4 to 4.8 selected from phosphoric acid, citric acid and ascorbic acid; j. Autoclaved at 110 °C k. When mixed with beer samples in a ratio of 1:10 to 1:20, this produces a pH value of the mixture of 4.4 to 4.8 and thus enables the growth or suppression of certain germs in the sample.