JP2008187972A - Method for measuring viable cell count of ice cream containing lactobacillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure a viable cell count other than that of lactobacillus, presenting as mixed in an ice cream containing lactobacillus, simply and quickly. <P>SOLUTION: A sample for measurement is prepared by diluting the ice cream containing the lactobacillus by 500 to 10,000 folds. The sample for measurement is cultured by a standard agar medium added with bromcresol purple. The bromcresol purple is added by 0.01 to 0.04 g per 1,000 mL standard agar medium. A mix dilution method is used for the culturing. Since the growth of the lactobacillus is inhibited by the restricted nutrition, etc., caused by the dilution, practically, it is possible to measure the viable cell count simply and quickly by a sufficient detection accuracy. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for measuring the number of viable bacteria other than lactic acid bacteria mixed in a frozen dessert containing live lactic acid bacteria, and more particularly to a method for measuring the number of viable bacteria suitable for ice creams to which lactic acid bacteria are added.

  Speaking of frozen desserts containing live lactic acid bacteria, for example, frozen yogurt. Lactic acid bacteria have attracted attention again in recent years because of their various health promotion effects such as intestinal regulation.

  On the other hand, ice cream is a representative example of cold confectionery. According to related laws and regulations, ice cream is classified into ice cream, ice milk, and lacto ice according to the difference in milk solid content and milk fat content, and these are defined as ice creams. Recently, a new frozen dessert in which lactic acid bacteria are added to the ice cream has been proposed. This ice cream containing lactic acid bacteria is not only a function of lactic acid bacteria added to ice creams, but also a refreshing aftertaste of fermented milk. Flavor is realized.

  By the way, since the component standards of these frozen desserts are stipulated by related laws and regulations, manufacturers that manufacture frozen desserts need to constantly measure and inspect that the standards are satisfied. One of the standards is a standard item for the number of bacteria (viable bacteria count, live bacteria count).

  However, since the number of bacteria is an indicator of bacterial contamination, when the test target includes lactic acid bacteria, the number of bacteria excluding lactic acid bacteria must be used as the number of bacteria. Non-patent documents 1 and 2 are examples of methods for measuring the number of viable bacteria when lactic acid bacteria are included in the test target.

“Food Sanitation Inspection Guidelines Microorganism Edition 2004”, Japan Food Sanitation Association, June 30, 2004, p. 579-580 "Butter, fermented milks and fresh chees-Enumeration of controlling microorganisms-Colony-counting technique at 30 ° C", ISO 13559: 2002 (E) (IDF 153: 2002)

  However, when the test object contains a large amount of lactic acid bacteria, such as frozen yogurt, it is difficult to measure the number of viable bacteria except for the lactic acid bacteria, and a sufficient measurement method has not been established.

  For example, it is conceivable to measure the number of general bacteria according to a standard method using a standard agar medium, and select lactic acid bacteria and general bacteria from the shape of the colony, etc., but if the test object contains a large amount of lactic acid bacteria, the lactic acid bacteria It is overwhelmingly more than that, requires experience to distinguish between them, lacks accuracy, and is not practical. In addition, a method in which a medium for measuring lactic acid bacteria is used in combination with this standard agar medium is also conceivable. In other words, the total number of bacteria including lactic acid bacteria and the number of lactic acid bacteria are measured and calculated by subtracting the number of lactic acid bacteria from the total number of bacteria, but this is too laborious and also lacks practicality. .

  Therefore, the present inventors have provided penicillin G potassium-added glucose as described in “Method for measuring the number of bacteria (viable cell count, but excluding lactic acid bacteria) of powdered soft drinks added with lactic acid bacteria” in Non-Patent Document 1. Attempts were made to apply agar medium or 4% sodium chloride-containing BCP-added plate count agar medium, but depending on the test subject, colonization of lactic acid bacteria was observed, and only general bacteria could not be selectively detected. It was.

  Further, in the previous Non-Patent Document 2, an international standard “Measurement Method of Contaminated Bacteria such as Fermented Milk” (hereinafter, simply referred to as “IDF153 Method”) is shown. In addition, since the culture condition requires a period of 30 ° C. and 72 hours, it is difficult to handle in quality control sites that require accuracy and speed. In addition, in this method, it is indispensable to add a selective agent to the medium, which requires a complicated operation such as selection of the selective agent and preparation of the medium, and is disadvantageous in that it lacks versatility.

  Therefore, in view of the above problems, the present invention measures the number of viable bacteria other than lactic acid bacteria mixed in a frozen dessert containing a large amount of live lactic acid bacteria, such as frozen yogurt and ice cream containing lactic acid bacteria, in a simple and rapid manner. It aims to provide a possible method.

  The present invention according to claim 1 is a method for measuring the number of viable bacteria in a frozen confectionery containing lactic acid bacteria, wherein the number of bacteria other than the lactic acid bacteria mixed in the frozen confectionery containing lactic acid bacteria is measured. And in order to solve the said subject, the process which prepares a measurement sample by diluting the said frozen dessert 500 times -10000 times, and the said measurement sample are culture | cultivated on the standard agar medium to which bromcresol purple (BCP) was added. And a step of counting the number of colonies detected after culturing.

  The present invention according to claim 2 is a method for measuring the number of viable bacteria of the frozen confectionery according to claim 1, wherein 0.01 to 0.04 g of bromcresol purple is added per 1000 ml of a standard agar medium. It is characterized by being.

  The present invention described in claim 3 is a method for measuring the viable cell count of the frozen confectionery containing lactic acid bacteria according to claim 1 or 2, characterized by culturing using a pour method.

  The present invention described in claim 4 is a method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria according to any one of claims 1 to 3, wherein the frozen dessert is an ice cream to which lactic acid bacteria are added. It is characterized by including.

  The viable count of frozen confectionery containing a large amount of live lactic acid bacteria can be measured simply and quickly with sufficient detection accuracy in practice.

  The present invention is a measurement method for measuring the number of bacteria other than lactic acid bacteria (viable bacteria number) mixed in a frozen dessert containing lactic acid bacteria. And in the measurement process, the step of preparing the measurement sample by diluting the frozen dessert to be measured 500 times to 10000 times, the step of culturing the measurement sample in a standard agar medium, and the number of colonies detected after the culture And a step of counting. The best mode for carrying out the present invention will be described below with reference to the drawings.

  In the present invention, a lactic acid bacterium is a bacterium contained in fermented milk typified by yoghurt, for example, and is a general term for a group of bacteria that produce a large amount of lactic acid. Bacteriologically, it belongs to the classifications such as Lactobacillus genus, Lactococcus genus and Streptococcus genus. There are many types of lactic acid bacteria, but in the present invention, the type of lactic acid bacteria is not particularly limited. However, among them, it is suitable when a lactic acid bacterium as a starter used for producing fermented milk, for example, lactic acid cocci (Streptococcus thermophilus), lactobacilli (Lactobacillus bulgaricus), or the like is included. In particular, lactic acid cocci are easily detected together with general bacteria among lactic acid bacteria, but their growth can be effectively suppressed.

  The measurement object of the present invention is a frozen dessert containing a large amount of live lactic acid bacteria such as frozen yogurt. Ice creams to which lactic acid bacteria have been added separately are also included in the measurement target. That is, in the present invention, a confectionery containing a large amount of live lactic acid bacteria, and a cold confectionery (frozen confectionery) handled in a frozen state during distribution or storage is used as a measurement target.

Since frozen desserts are distributed under freezing, general bacteria cannot grow due to temperature conditions after production. Therefore, if the number of viable bacteria is inspected after production to confirm that it is within the standard, then there is no need to worry about the increase in the number of bacteria. And, for example, looking at the specification of the number of bacteria in the previous ice cream, ice cream is 100,000 (1 × 10 ⁵ ) cfu (colony forming unit) or less per gram, ice milk and lacto ice are 50 or less per gram. 000 (5 × 10 ⁴ ) cfu or less, and even if the detection lower limit number of bacteria is relatively high, it can be determined whether or not the standard is satisfied, and the measurement conditions are advantageous.

However, fermented milk such as yogurt usually contains about 10 ⁷ cfu lactic acid bacteria per gram. For this reason, it is not easy to selectively measure only the general bacteria present in the mixture.

  However, lactic acid bacteria tend to be more auxotrophic than other general bacteria. In view of the characteristics of frozen confectionery, the present inventors have sufficiently increased the dilution rate at the time of measurement to maintain the nutrient source derived from frozen confectionery, that is, the nutrient source that is considered effective for the growth of lactic acid bacteria in the medium. We thought that only general bacteria could be selectively measured if the amount of incorporation was reduced. As a result of verification by experiments, a practical measurement method has been obtained.

  In other words, in the present invention, the measurement sample is prepared by first diluting the frozen confectionery to be measured 500 times to 10000 times, preferably 500 times to 5000 times considering the standard and inspection accuracy. Since it can cope with simple repetition of 10 times dilution, More preferably, it dilutes 1000 times. By doing so, first, the growth of lactic acid bacteria can be effectively suppressed by nutritional restriction, and even if a large amount of lactic acid bacteria are included, only general bacteria mixed there can be selected and measured. It becomes possible. However, in some cases, yellowing or the like is not observed in the medium and general bacteria can be measured. In such a case, the dilution factor is not necessarily limited. The dilution factor does not require strict accuracy. These numerical values include an acceptable front-rear width.

  FIG. 1 shows an outline of the measurement method of the present invention. As shown to (a) of FIG. 1, 1 g was extract | collected from the frozen dessert containing the lactic acid bacteria which are measurement objects in aseptic environment, such as a clean bench, and the physiological saline (salt concentration 0.85%) was contained. Repeat dilutions in sterile tubes and dilute stepwise up to 1000 times. And the solution which diluted and prepared frozen confectionery 1000 times is set as the measurement sample S.

  The culture is performed using a medium suitable for culturing general bacteria, that is, a standard agar medium for measuring general bacteria commonly used.

  Bromocresol purple (BCP) is preferably added to the standard agar medium. Specifically, 0.01 to 0.04 g of BCP is added per 1000 ml of standard agar medium. Preferably, 0.02 to 0.03 g of BCP is added. BCP is a PH indicator composed of a blue pigment. As the medium, for example, it is used as a BCP-added plate agar medium which is an official medium for measuring lactic acid bacteria. In this embodiment, 0.025 g of BCP is added per 1000 ml of standard agar medium.

  Here, the composition of each medium is shown in FIG. FIG. 2 shows a comparison between a standard agar medium, a BCP-added plate agar medium, and a medium obtained by adding BCP to the standard agar medium (hereinafter simply referred to as Blue-SMA). Each medium has the same basic composition of peptone, yeast extract, glucose and agar. The difference with respect to the standard agar medium is that Blue-SMA contains BCP, and the BCP-added plate agar medium further contains tween 80 (registered trademark) and L-cysteine.

  Tween 80 and L-cysteine are nutrient sources added in accordance with the auxotrophy of lactic acid bacteria. Therefore, the standard agar medium and Blue-SMA not containing this can suppress the growth of lactic acid bacteria accordingly.

  BCP is used in a BCP-added plate agar medium to facilitate the visual detection of lactic acid bacteria by utilizing the fact that the periphery of the colony is yellowed by lactic acid produced by lactic acid bacteria. On the other hand, in the present invention, by utilizing the fact that the periphery of colonies of general bacteria that produce some acid turns yellow, it is possible to easily detect general bacteria visually or to distinguish colonies detected with increased contrast. Used for facilitating.

  Furthermore, it was found that by adding BCP to the standard agar medium in the course of the experiment, the effect of suppressing the growth of lactic acid bacteria was recognized, and the BCP in the present invention can also contribute to the effect of suppressing the growth of lactic acid bacteria. Yes. The experimental results are shown in FIG. FIG. 3 shows the result of measuring the number of viable bacteria using a certain frozen dessert containing lactic acid bacteria as a sample. In this case, under the same predetermined conditions, a comparison experiment was performed using a standard agar medium (manufactured by MERCK) with a different dilution ratio of the sample (S) and Blue-SMA prepared by adding BCP to the standard agar medium. (N = 2, measured twice for each sample).

As shown in FIG. 3, in a standard agar medium not containing BCP, 10-fold dilution is indistinguishable, and in 100-fold dilution, too many colonies cannot be counted (TNTC; Too Numerous To Count), 1000 Bacterial growth was observed even at double dilution (for example, 2.9 × 10 ⁵ cfu / g). On the other hand, in Blue-SMA to which BCP was added, yellowing was observed up to 100-fold dilution, but colony formation was not observed at 1000-fold dilution. That is, it is considered that the growth of lactic acid bacteria could be effectively suppressed by the nutritional restriction by dilution and the synergistic effect of addition of BCP.

  Next, the culture operation will be described. Prior to the operation, a sufficient amount of the culture medium is prepared in advance and aliquoted and sterilized. And the required amount of a culture medium is heated at the time of operation, and it makes it liquid state. By doing so, you can work together and work efficiently. The liquid medium (medium that has been heated and dissolved after sterilization) is kept at a temperature that does not affect the bacteria as it is, for example, around 40 ° C.

  Then, as shown in FIG. 1B, 1 ml of the prepared measurement sample S is poured into a sterilized petri dish P together with an appropriate amount of medium (Blue-SMA), and immediately solidified while mixing. As shown in FIG. 1C, the petri dish P in which the medium is solidified is stored in the thermostat T in a state where the medium is faced down and cultured under a predetermined condition (pour method). Since the operation is simply mixing the culture medium and the measurement sample, even if a large number of tests are required, the operation can be performed easily and quickly.

  In addition, there is a method (smearing method) in which a culture plate is solidified in a Petri dish P in advance and a measurement sample is dropped on the plate and spread on a flat plate with a conrad rod or the like (smearing method). An attempt to lower the lower limit of detection number of bacteria requires an increase in the number of petri dishes, which necessitates preparation and operation, and is inferior to the pour method in practical terms.

  As for the culture conditions, when some low-temperature bacteria and mesophilic bacteria are targeted, the culture is performed at 30 ° C. for 48 hours. In the case of targeting mesophilic bacteria, the cells are cultured at 35 ° C. for 48 hours. However, the culture conditions can be appropriately prepared as necessary. Moreover, the numerical value of the culture condition shown here is an optimum condition and does not require strict accuracy. These numerical values include an acceptable front-rear width.

  After culturing under predetermined conditions, colonies formed on the solidified medium are counted visually. Based on the obtained count value, the number of viable bacteria per unit amount of the frozen dessert is calculated from the dilution rate. Since lactic acid bacteria are suppressed in growth and do not form colonies, they can be measured with confidence.

  At this time, if a predetermined amount of BCP is added, the contrast increases, so that colonies can be easily identified. That is, in the case of the pour method, colonies tend to be minute because colonies are formed inside the medium, but even in this case, the colonies can be clearly seen by contrast.

  As described above, according to the measuring method of the present invention, a frozen confectionery containing a large amount of lactic acid bacteria, which has been time-consuming and time-consuming, has been required only by diluting the frozen confectionery by a predetermined amount by devising a conventionally used medium. This makes it possible to easily and quickly carry out the bacterial test.

Of course, the number of detection lower limit bacteria is increased by the amount of dilution, but since it is intended for frozen desserts, it is sufficient to determine whether or not the standard is satisfied, considering the standard. For example, in the case of ice cream, the standard standard is 100,000 (1 × 10 ⁵ ) cfu / ml or less, so whether or not 10 colonies are detected in the petri dish even if diluted 10,000 times. It can be judged whether or not the standard is satisfied. In the case of lacto ice or ice milk, the standard standard is 50,000 (5 × 10 ⁴ ) cfu / ml or less. Whether 10 standard colonies are detected or not can be judged whether or not the standard is satisfied.

{First Example}
As a first example, an experiment in which the measurement method of the present invention (hereinafter referred to as the Blue-SMA method) is compared with the measurement method by the IDF153 method described above is shown.

As an object to be measured, ice cream containing lactic acid bacteria using 40% fermented milk was used. This contains 12.5% non-fat milk solids, 13.5% milk fat, and about 10 ⁷ cfu / g lactic acid bacteria. The lactic acid bacteria contained are starter lactic acid bacteria used in the production of fermented milk. Specifically, Streptococcus thermophilus OLS3290 strain (FERM P-19638) and Lactobacillus bulgaricus OLL1073R-1 strain (FERM BP-10741). A total of 17 samples were measured for each lot of ice cream containing lactic acid bacteria.

  As the medium, the medium (Blue-SMA, manufactured by MERCK) obtained by adding BCP to the standard agar medium described above was used. The culture temperature was 30 ° C. and 35 ° C., and the culture period was 48 hours.

The measurement sample, the lactic acid bacteria-containing ice cream to be measured, sterile saline 10-fold (salinity 0.85%) ⁽¹⁰ -1), 100-fold ^(10-2), 1000-fold (10 ^- What was diluted in ³ ) was used. At 35 ° C., in addition to that, the sample was diluted 10,000 times (10 ⁻⁴ ) and 100,000 times (10 ⁻⁵ ), and each was used as a measurement sample.

  1 ml of each measurement sample obtained was mixed with an appropriate amount of Blue-SMA and a sterilized petri dish to be solidified, and then cultured at each temperature for 48 hours, and the number of colonies detected was counted visually.

  As a comparison object, measurement was performed according to the IDF153 method for each of the 17 samples simultaneously with the above measurement. Specifically, a medium plate was prepared in a sterilized petri dish using a predetermined medium in the same method. Then, after 0.1 ml of each measurement sample diluted 100 times to 10000 times above was smeared on a medium plate, it was cultured at 30 ° C. for 72 hours, and the number of colonies detected was counted visually.

  In addition, the number of lactic acid bacteria contained in each sample was also measured according to a ministerial ordinance such as milk.

In the Blue-SMA method, yellowing over the entire medium was observed at 10-fold dilution. In addition, discoloration of the medium was observed even at 100-fold dilution. Therefore, these were judged to be affected by lactic acid bacteria and excluded from the measurement target. On the other hand, when the dilution was 1000 times or more, no color change was observed in the medium. Therefore, a measurement sample having a 1000-fold dilution or more was employed, and the number of colonies formed in the medium of each measurement sample was counted. The counting results are shown in FIG. As shown in FIG. 4, about 10 ⁶ to 10 ⁸ cfu / g lactic acid bacteria were contained in any of the 17 ice cream samples containing lactic acid bacteria. And based on the count result equivalent to 1000-fold dilution, the number of viable bacteria per 1 g of frozen dessert was calculated from the dilution rate. The calculation results are shown in the table of FIG. 5 and the graphs of FIGS. In this case, the detection limit is 1000 cfu / g (<1000).

As shown in FIG. 4 and FIG. 5, in the Blue-SMA method, the maximum viable count of 37000 cfu / g was measured from no detection. In FIG. Although detection is recognized in a part that should not be detected originally from the dilution factor, such as 100000 times (10 ⁻⁵ ) of 11, this is considered to be an operational error associated with the bacterial test. Therefore, it is difficult to determine the presence or absence of detection with respect to the standard because the accuracy is low even with a few count values even qualitatively. Therefore, in this example, the 1000-fold dilution that can be compared with a certain number of detection values is evaluated. As shown in FIG. 5, the results of the 1000-fold diluted measurement sample were 9 samples at 30 ° C., 6 samples at 1000 cfu / g level, and 2 samples at 10000 cfu / g level at 30 ° C. Similarly, at 35 ° C., there were 7 samples without detection, 4 samples at 1000 cfu / g level, and 6 samples at 10000 cfu / g level.

In contrast, in the IDF153 method, viable cell counts of up to 27000 cfu / g were measured from no detection. The results for the measurement sample (10 ⁻² ) corresponding to 1000-fold dilution were 8 samples without detection, 6 samples at 1000 cfu / g level, and 3 samples at 10000 cfu / g level.

  FIG. 6 and FIG. 7 are graphs comparing the measurement results of the viable cell count of each sample in the measurement sample corresponding to 1000-fold dilution of frozen dessert with the Blue-SMA method and the IDF 153 method from the results shown in FIG. It is. FIG. 6 shows the results when the culture temperature is 30 ° C., and FIG. 7 shows the results when the culture temperature is 35 ° C. As shown in each graph, the Blue-SMA method showed almost the same detection tendency as the IDF153 method in both cases of 30 ° C. and 35 ° C. In particular, a slight difference was observed at a level where the number of viable bacteria was small and the error was large, but this tendency was recognized when the number of viable bacteria increased.

  Therefore, it can be seen that the Blue-SMA method has the same detection accuracy as the IDF153 method.

{Second Example}
As a second example, an experiment in which the influence of the added amount of BCP is confirmed is shown. In this experiment, the number of viable bacteria was measured using a certain frozen dessert containing lactic acid bacteria as a sample and changing only the amount of Blue-SMA BCP added.

  Blue-SMA was prepared in which the amount of BCP added per 1000 ml of standard agar medium was 0.020 g, 0.025 g, and 0.030 g. Then, the number of viable bacteria was measured for each sample (10 times to 1000 times) with different dilution ratios using the prepared Blue-SMA (N = 2, measured twice for each sample). The culture conditions were all cultivated at a temperature of 35 ° C. for 48 hours, and the operation and others were performed under the same conditions. The number of lactic acid bacteria contained in each sample was also measured according to an ordinance such as milk.

The result is shown in FIG. As shown in FIG. 8, yellowing was observed at the 10-fold and 100-fold dilutions regardless of the amount of BCP added, and the effect of lactic acid bacteria was observed, but yellowing was observed at the 500-fold dilution and 1000-fold dilutions. In addition, no colonies were observed. Although not shown in the drawings, about 10 ⁷ to 10 ⁸ cfu / g of lactic acid bacteria were observed in each measured sample. Therefore, it was found that as long as at least the above-mentioned amount of BCP was added, there was no difference in the effect of suppressing the growth of lactic acid bacteria, and the measurement could be performed reliably and stably.

  As described above, according to the present invention, the number of viable bacteria in frozen confectionery containing a large amount of live lactic acid bacteria, which could not be measured without taking time and effort, such as the IDF153 method, has been practically sufficient. Therefore, it becomes possible to measure easily and quickly.

It is a figure explaining the outline of the measuring method of the present invention. It is a figure which shows the composition of various culture media. It is a figure showing the experimental result which shows the growth inhibitory effect of lactic acid bacteria by BCP addition. It is a figure which shows the experimental result of 1st Example. It is a figure which shows the experimental result of 1st Example. It is a graph which shows the experimental result of 1st Example. It is a graph which shows the experimental result of 1st Example. It is a figure which shows the experimental result of 2nd Example.

Explanation of symbols

S Measurement sample P Petri dish T Incubator

Claims (4)

A method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria, which measures the number of bacteria other than the lactic acid bacteria mixed in the frozen dessert containing lactic acid bacteria,
A step of diluting the frozen dessert 500 times to 10000 times to prepare a measurement sample;
Culturing the measurement sample on a standard agar medium supplemented with bromcresol purple;
Counting the number of colonies detected after culturing;
A method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria. A method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria according to claim 1,
A method for measuring the viable cell count of a frozen confectionery containing lactic acid bacteria, wherein 0.01 to 0.04 g of bromcresol purple is added per 1000 ml of a standard agar medium. A method for measuring the number of viable bacteria of the frozen confectionery according to claim 1 or 2,
A method for measuring the number of viable bacteria of a frozen dessert containing lactic acid bacteria, characterized by culturing using a pour method. A method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria according to any one of claims 1 to 3,
The method for measuring the number of viable bacteria in a frozen dessert containing lactic acid bacteria, wherein the frozen dessert includes ice cream to which lactic acid bacteria are added.

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