Liquid cell culture media (nutrient solution) for the detection of viable microorganisms with obligatory product damage potential in non-alcoholic 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 a minimum of 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 4.0.

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 have no direct impact on the product. A negative change from these germs is not to be expected, as they are not capable of reproduction under the product's properties. Examples would be coliform bacteria / E. coli, Legionella, bacilli or enterococci. These cannot grow in a soft drink that has an oxygen-free atmosphere, acids or an acidic pH value (below 4.0) and inhibitors, but can still be present and thus serve as an indicator of hygiene deficiencies. These germs are also referred to as potentially harmful, as 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 or ingredients, thus resulting in or causing changes to or damage to the products. 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 would be, for example, acid-tolerant bacteria such as lactic acid bacteria or fermentable yeasts.

[0005] The following detection procedure is currently used for detection in the in-house laboratories of beverage manufacturers and contract laboratories: Liquid enrichment of beverage samples. This involves filling a specific volume of sample (e.g., 1-200 ml) in a container such as a laboratory bottle with liquid nutrient solution in the same ratio as, or more than, the sample. These nutrient solutions often have similar compositional properties to the finished beverage, allowing growth of only specific microorganisms using specific selective factors and inhibitors. This test should then detect, if possible, only those microorganisms that are harmful to the product. This is done by multiplication and subsequent visual evaluation of the increase in turbidity, or microscopically, or via 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 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 beverage sample (pH value usually between 3.5 - 3.9) is mixed with a nutrient medium (pH value between 5.0-7.9), the pH value is above 4.0 and thus higher than that of the finished beverage.

[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] This poses the following problem for analyses using finished beverages. A beverage sample is over-diluted by adding large volumes of a normal nutrient solution. Therefore, highly concentrated nutrient solutions with a suitable pH value have been developed that are so similar to the final beverage that only minimal addition volumes are necessary to enable the growth of obligately harmful bacteria and subsequently provide detection. At the same time, the beverage is altered as little as possible, and the product's own inhibitors are barely reduced. For example, in a lemonade, adding 2 to 5 vol% of concentrated nutrient solution can achieve the usual visual detection of obligately harmful bacteria without the addition drastically altering the beverage.

[0013] 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 as much as possible and can thus be virtually eliminated.

[0014] This also applies to specialty beverages that have less common properties, such as a very low pH value. Likewise, beverages that contain other inhibitors, such as preservatives (e.g., benzoates, sorbates, sulfur, hop bittering compounds, oils, extracts, aromatics, etc.), which should be altered as little as possible.

[0015] 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 of less than 4.

0. [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 bythat 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 4.0, preferably less than 3.8; particularly preferably less than 3.

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 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 3.5 to 3.9, selected from phosphoric acid, citric acid and ascorbic acid; h. Autoclaved at 110 °C; i. When mixed with beverage samples in a ratio of 1:10 to 1:20, 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.