Liquid cell culture media (nutrient solution) for the detection of viable microorganisms with obligatory product damage potential for non-alcoholic beverages, analyzed in water 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 color indicators; and characterized by the fact that the nutrient solution has a pH value of less than 4.5.

Description

[0001] As part of quality assurance, food and beverage manufacturers must monitor their raw materials, ingredients, intermediate products, and finished beverages for microbiological contamination, among other things. A fundamental distinction is made here between so-called hygiene indicators and germs that are obligatory for product deterioration.

[0002] Hygiene indicators are microorganisms that can be detected in a sample and thus serve as evidence of entry into the process, but will have no direct impact on the finished product. Negative changes caused by these germs are not to be expected, as they are unable to reproduce under the product's characteristics. Examples would be coliform bacteria / E. coli, Legionella, bacilli, or enterococci. These cannot grow in a soft drink that, for example, has an oxygen-free atmosphere, acids or an acidic pH (below 4.0), and inhibitors, but may still be present and thus serve as an indicator of hygiene deficiencies. These germs 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 prohibited.

[0003] Obligate product-damaging bacteria are those that are usually capable of multiplying in the finished beverage and thus cause or result in a change in or damage to the product. Such bacteria grow under the characteristics present in the sample, despite the presence of inhibitors.

[0004] For example, in soft drink production, the focus of quality assurance is on so-called beverage-spoiling bacteria and yeasts that are capable of multiplying under the prevailing conditions. These are germs that thrive in the presence of many organic acids or a low pH value below 4.0 and CO 2 (no oxygen) can still grow. These include, for example, acid-tolerant bacteria such as lactic acid bacteria or fermentable yeasts.

[0005] The following procedure is currently used for detection in the in-house laboratories of beverage manufacturers and contract laboratories: Liquid enrichment of water samples. This involves filling a specific volume of the sample (e.g., 1-200 ml) in a container, such as a laboratory bottle, with liquid nutrient solution. These nutrient solutions often have similar compositional properties to the finished beverage, allowing specific microorganisms to grow using specific selective factors and inhibitors. This test is intended to detect microorganisms that are potentially harmful to the product. This is done by multiplication and subsequent visual evaluation of the increase in turbidity, microscopically, or via DNA analysis such as PCR.

[0006] In this case, however, the nutrient solutions, and especially the mixtures of nutrient solution and water, are not comparable to the final drink. The main difference lies in the pH value. pH is a crucial value for growth, and since it is also a logarithmic concentration value, it is particularly important.

[0007] For example, if a water sample (typically between 6.0 and 8.0) is mixed with a nutrient medium (pH between 5.0 and 7.9), the pH will be well above 5.0 and thus higher than that of the finished beverage. However, it is often essential to test water samples for germs that are inherently harmful to products, as these waters are used for cleaning purposes with product contact or even as an ingredient.

[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 Beverage-harming germs 5,0 Miscellaneous MRS Lactic acid bacteria 6,2 Doehler Trans-fast Beverage-harming germs 6,1 Difco Micro Inoculum Broth Beer-spoiling germs 6,7 Doemens DS Select Beer-spoiling germs 5,4

[0009] Beverages from the beverage industry, such as lemonades, soft drinks, juices, spritzers, etc., typically have a pH value below 4.0. This poses the problem that either bacteria contained in the sample that grow at pH values ​​below 4.0 cannot be detected, or bacteria that would not be able to reproduce in the final beverage grow. In both cases, the analysis must be considered incorrect.

[0010] In the real-life process of industrial laboratories, this regularly leads to deviations, product damage, recalls, and even potential consumer harm. In the author's opinion, there is no evidence available on the market that adequately takes this fact into account.

[0011] Thus, through extensive research, many trials and tests, various recipes and nutrient solutions have been developed that can solve this problem while adhering to certain specifications.

[0012] Invention for water sampling in the soft drinks industry.

[0013] For samples, especially water samples, specific, highly concentrated nutrient solutions with a suitable pH value have been developed. These nutrient solutions have special properties due to the high concentration of organic acids and dyes with pH indicators, as well as growth factors consisting of carbon sources, nitrogen sources, fatty acid sources, and minerals. When this concentrated nutrient solution is then mixed with water at a ratio of approximately 1:3 to 1:5, the finished mixture exhibits properties very similar to soft drinks.

[0014] If, for example, a standard "product water" (200 ml) is mixed with 50 ml of this new nutrient solution, the mixture results in a pH value of approximately 3.6 to 3.9. This pH value can be visually indicated by a color change of the indicators, making it easier for the user to correctly mix and adjust the desired pH value. This pH value then corresponds to a common European soft drink such as lemonade, fruit juice spritzers, soft drinks, juice, or a non-alcoholic beer mix, thus replicating this type of beverage as closely as possible. This enables the growth of necessarily harmful germs and, consequently, ensures correct detection.

[0015] Surprisingly, only germs that are actually or necessarily harmful to the product can grow. False results caused by the growth of "hygiene indicators" or potentially harmful germs are suppressed to the greatest possible extent and can thus be virtually eliminated.

[0016] The goal is to accurately represent the actual product damage without the influence of hygiene indicators by adding specific nutrient solutions. Processing should also be simplified by adding a high amount 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 color indicators; as well as characterized by that the nutrient solution has a pH value below 4.

5. [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, additionally comprising color indicators which make the mixing or adjustment of the desired mixture visually apparent (color change) and thus simplify the use. [4] Nutrient solution according to one of the preceding claims, characterized bythat as the germs grow or multiply, they can be removed from the sample and further analyzed. [5] 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. [6] Nutrient solution according to one of the preceding claims, characterized by that it has a pH value of less than 4.5, preferably less than 4.0; particularly preferably less than 3.

5. [7] Nutrient solution according to one of the preceding claims, characterized bythat 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. [8] 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 and glucose; 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. dyes selected from bromophenol blue, bromophenol red, methylene blue; f. Demineralized water; g. Acids to achieve a pH of 3.0 to 3.5, selected from phosphoric acid, citric acid and ascorbic acid; h. Autoclaved at 110 °C; i. When mixed with water samples in a ratio of 1:3 to 1:5, this produces a pH value of the mixture of 3.5 to 3.9 and thus enables the growth or suppression of certain germs in the sample.