JPH04349878A - Production of starter - Google Patents

Abstract

PURPOSE:To provide a starter high in bacterial concentration and activity, capable of determining the time for culture cessation quickly and accurately. CONSTITUTION:In producing a starter, the ATP luminescence level for a starter culture solution is measured with the elapse of time at regular intervals, and the culture is discontinued when the logarithmic ratio of the measurement to the preceding measurement comes within the range 1.06-0.98.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a starter. More specifically, in the production of a starter, the present invention quickly determines when to stop the culture.
The present invention relates to a method for producing a starter with high microbial concentration and activity by accurately determining the concentration and activity of microorganisms.

0002

BACKGROUND OF THE INVENTION Starters have been widely used in the production of yogurt, cheese, fermented butter, and lactic acid bacteria beverages. Such starters have a high microbial concentration and activity when the microorganisms used are in the late logarithmic growth phase or early stationary phase, and are considered optimal for producing the above products.

[0003] Generally, as a method for determining the phase of microbial growth, there is a plate counting method in which the number of bacteria in a culture solution is measured and changes in the number of bacteria over time are examined. In addition, the method for measuring lactic acid bacteria in fermented milk and lactic acid bacteria drinks (official method, "Ministerial Ordinance on Ingredient Standards for Milk and Dairy Products" [December 27, 1950)
Japan, Ministry of Health and Welfare Ordinance No. 52] also uses the plate counting method. As an indirect method, the correlation between the number of bacteria and the acidity of the culture solution, the correlation between the number of bacteria and the pH of the culture solution, the correlation between the number of bacteria and the culture time, etc. are investigated in advance, and the acidity and pH of the sample culture solution are investigated. There are known methods for estimating the number of bacteria or the phase of growth of microorganisms from the measured value or culture time.

On the other hand, intracellular A of microorganisms present in the sample
A method in which the amount of TP is measured from the amount of light generated by the chemical reaction between ATP and luciferin/luciferase and converted to the number of bacteria. A method (hereinafter referred to as ATP method 1) that measures the amount of ATP emitted from microorganisms in a sample and calculates the number of bacteria in the sample has been developed in recent years, and this method has been used to calculate the number of microorganisms in raw milk and yogurt. A rapid detection method has been reported (Journal of Food Hygiene, Vol. 25, No. 2, pp. 193-197, 1984). Furthermore, the ATP platform test and the ΔATP test method (hereinafter referred to as the "ΔATP test method" as ATP method 2) have been reported to measure the microbial quality of raw milk [Netherlands Milk and Dairy Journal (Netherlands Milk and Dairy Journal)].
thelands Milk and Dairy
Journal), Volume 42, Pages 173-182,
1988], and a method with improved sensitivity for measuring the number of bacteria in milk samples using the bioluminescence method (hereinafter referred to as ATP method 3) was reported [Journal of Food
Protection (Journal of Food P
rotation), Volume 51, No. 12, 949-
954 pages, 1988].

[0005]

[Problems to be Solved by the Invention] However, among the above-mentioned conventional techniques, the plate counting method requires 12 to 24 hours (72 hours in the official method) to obtain the result of bacterial count measurement. By the time the growth phase of the microorganism is determined, the culture solution has already passed that phase and cannot be used as an optimal starter. The method of indirectly estimating the number of bacteria or the phase of growth from the acidity and pH values of the culture solution is not suitable for accurately determining the number of bacteria or the phase of growth because the correlation between acidity and pH and the number of bacteria is low. However, the method of estimating from culture time is also inappropriate for estimating the phase of microbial growth because the growth curve fluctuates due to subtle differences in microbial activity, culture temperature, and medium components. Furthermore, when estimating from acidity, pH, and culture time, the correlation varies depending on conditions such as the type of medium used and the type of microorganism, so it is inconvenient that the correlation must be investigated in advance every time the conditions change. There is.

On the other hand, ATP method 1 is a method for calculating the number of bacteria in a sample, but since the amount of ATP in one microorganism cell differs depending on the type of microorganism, the average amount of ATP in one cell for each bacterial type is calculated in advance. Since it is necessary to measure the amount of ATP and then measure the amount of ATP derived from microorganisms in the sample to calculate the number of bacteria, it is too complicated and time-consuming as a means of determining the phase of microbial growth. It is difficult to apply to industrial production of starter.

[0007] ATP method 2 calculates the number of bacteria in a sample using a regression equation from the difference between the luminometer readings obtained by changing a part of the analysis process of ATP method 1 and the luminometer readings according to ATP method 1. The microbial concentration measurement level of ATP method 1 is 106/ml, while the ATP method
Method 2 is said to be able to measure bacterial concentration levels up to 105/ml. However, this report only aims to sensitively measure the number of microorganisms present in raw milk, and cannot determine the phase of microbial growth.

In ATP method 3, a large amount of sample is used, treated with a surfactant, trypsin treatment, and filtered with a Nucleopol polycarbonate filter (0.4 μm). is a method for calculating the number of bacteria, and the microorganism concentration measurement level is 1.
05/ml. However, this report is also only aimed at sensitively measuring microorganisms in raw milk, and like ATP method 2, it cannot determine the phase of microorganism growth.

[0009] The present invention was made in view of the above-mentioned circumstances, and eliminates the drawbacks of the conventional starter production method. The present invention aims to provide a method for producing a starter with a high concentration of microorganisms and the highest activity by quickly and accurately determining when to stop culturing a starter culture solution.

0010

[Means for Solving the Problems] The present invention solves the above-mentioned problems by measuring the amount of ATP luminescence of a starter culture solution over time at regular intervals in the production of a starter, and measuring the amount of luminescence. Provided is a method for producing a starter, characterized in that culture is stopped when the ratio between the logarithm of the value and the logarithm of the previous measured value of luminescence amount is in the range of 1.06 to 0.98.

[0011] This invention will be explained in more detail below. The present invention can be applied to a method for producing a starter, that is, a conventional method for producing a starter using a commonly used culture medium and microorganisms (eg, bifidobacteria, lactobacilli, lactic acid cocci, yeast, etc.). To measure the amount of luminescence in the culture solution, known methods and conditions for measuring ATP using a chemical reaction between ATP and luciferin/luciferase can be applied, and commercially available reagent kits can be used.

[0012] In order to measure the amount of luminescence of a culture solution in the method of the present invention, samples of the culture solution are collected at regular time intervals, a reagent is mixed, and the amount of luminescence is read as a value on a luminometer. Since it only compares the logarithm of the values and does not measure the number of bacteria, it is necessary to prepare a calibration curve to know the absolute amount of ATP as in ATP method 1.
It is not necessary to measure the average amount of ATP per bacterial cell. Also, A
There is no need to perform analysis twice on the same sample in parallel as in TP method 2, and there is no need to create a regression equation as in ATP method 3. The measurement value of the culture liquid light amount is a certain time (eg, 6, 6, (eg, 6, 6, 6, (eg, 6
This is the integrated value of the amount of luminescence read with a luminometer.

[0013] The recommended time interval for measuring the amount of luminescence of the culture solution is 10 minutes or more and 40 minutes or less, particularly preferably 20 minutes to 30 minutes. The culture is stopped when the luminescence amount of the culture solution, that is, the ratio of the logarithm of the luminometer reading falls within the range of 1.06 to 0.98. This task for determining culture termination is particularly difficult because calculating the above ratio simply involves dividing the logarithm of the current luminometer reading by the logarithm of the previous luminometer reading. Rather, it is possible to know very easily and quickly whether the ratio has fallen within a predetermined range. Note that if the time interval between measurements is too short, the determination of the time to stop the culture may be inaccurate (for example, when measurements are taken continuously, the ratio is 1. 00); conversely, if the time interval between measurements is too long, there is a risk of losing the optimal timing as a starter;
A measurement interval of 0 minutes or more and 40 minutes or less is recommended.

As explained above, in the method of the present invention,
Without measuring the number of bacteria in the culture solution, using the growth curve of microorganisms, or calculating the number of bacteria using a regression equation,
By simply calculating the ratio of the logarithm of the luminescence amount of the culture solution (luminometer reading), it is possible to easily and accurately determine the appropriate time to stop the culture almost instantly (about 2 minutes from sample collection to the end of measurement). It is possible to produce starters with high microbial concentration and high activity. Furthermore, since the method of the present invention facilitates automatic measurement of the amount of luminescence of the culture solution, it is possible to use a recorder to graphically display changes in the measured value over time, or to calculate the logarithm and logarithm ratio of the amount of luminescence of the culture solution.
It is also possible to perform online process control for starter manufacturing using a controller in which the set increase rate is input.

[0015] Next, an example of a test conducted in order to determine a preferable range of the logarithmic ratio of the luminescence amount of the culture solution in the method of the present invention will be shown. Test Example A starter was cultured using a conventional method, and 3 hours after the start of culture.
Every 0 minutes, a sample for analysis and a sample for yogurt production (for inoculation) are collected, and the amount of luminescence of the culture solution is measured, the ratio of its logarithm is calculated, and yogurt production is performed, and the above ratio and yogurt production time are calculated. We investigated the relationship between

1. Starter culture method (1) Bacteria used:
Lactobacillus bulgaricus (manufactured by Hansen) and Streptococcus thermophilus (manufactured by Hansen)
. A mother starter was prepared and used in the same manner as described in Example 1. (2) Medium: Sterilized skim milk with non-fat milk solids content of 10% (weight) (3) Medium amount: 30 kg (4) Mother Starter inoculation amount: Lactobacillus bulgaricus mother starter 450g and Streptococcus thermophilus mother starter 450g
g(5) Culture temperature: 37°C 2. Measurement of the luminescence amount of the culture solution and calculation of the ratio (1) Sampling: Collect 20 μl of the culture solution from the culture using the method described above. (2) Reagent: ATP monitor kit (lytic reagent MI
CROLYSE and the luminescent reagent MICROZYME. (3) Measuring equipment: Luminometer LKB-Walla
c1251 (manufactured by LKB Wallac) (4) Measurement procedure: Measure the luminescence amount for 60 seconds and calculate the integrated value [millivolt x second (mV・s)]
] was sought.

(5) Calculation: Ratio = log (current integrated value)/log (last integrated value [30 minutes ago]) 3. Yogurt production method: Pasteurized skimmed milk with non-fat milk solids content of 10% (weight; the same applies hereinafter) 2
Add and mix 6g of the culture solution in the culture using the above method to 00g, leave it to stand at 37°C for 0 to 6 hours, and measure the time until the acidity of the mixture reaches 0.8% (fermentation completion time) did.

[0018] The timing of completion of fermentation of yogurt is usually determined based on acidity (0.8%), so this was followed. 4. Test Results The results of this test are shown in Table 1.

[0019]

[Table 1]

As a result of this test, the amount of luminescence of the culture solution (mV・s
) that the ratio of the logarithm of the current measurement value divided by the logarithm of the previous measurement value is 1.06 to 0.98. When a culture solution is used as a starter, yogurt can be obtained in an appropriate time. On the other hand, it was confirmed that when the above ratio exceeds 1.06 or is less than 0.98, it takes a long time to finish the yogurt and the hardness of the yogurt decreases. Therefore, the ratio of the logarithm of the current measurement value of the culture solution luminescence amount divided by the logarithm of the previous measurement value is 1.06 to 0.9.
By stopping the culture at a temperature of 8, particularly preferably 1.05 to 1.00, a highly active starter can be produced.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[0022]

【Example】

Example 1 Commercially available Lactobacillus bulgaricus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium, sterile skim milk was inoculated with 3% of the above culture, and incubated at 37°C for 20
After culturing for hours, a mother starter of Lactobacillus bulgaricus was prepared. Separately commercially available Streptococcus
Streptococcus thermophilus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium, 3% of the above culture was inoculated into sterile skim milk, and cultured at 37°C for 20 hours. A mother starter of Filus was prepared. 0.1 kg of the above Lactobacillus bulgaricus mother starter and 0.1 kg of Streptococcus thermophilus mother starter were added and mixed to 10 kg of sterilized skim milk with a non-fat milk solid content of 10%, and cultured at 37°C. During culture, the luminescence amount of the culture solution was measured every 30 minutes using the same method as in the test example, and the logarithm of the luminescence amount was calculated as 3.
The value (ratio) divided by the logarithm of the amount of light emitted 0 minutes before was calculated. After 4 hours and 30 minutes after starting the culture, the ratio reached 1.00, so the culture was stopped and about 10k of highly active symbiotic starter was added.
I got g. Example 2 Commercially available Lactobacillus bulgaricus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium. Sterilized skim milk was inoculated with 3% of the above culture, and incubated at 37°C for 14 days.
After culturing for hours, a mother starter of Lactobacillus bulgaricus was prepared. Separately commercially available Streptococcus
Streptococcus thermophilus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium, sterile skim milk was inoculated with 3% of the above culture, and cultured at 37°C for 14 hours. A mother starter of Filus was prepared. 0.45 kg of the above Lactobacillus bulgaricus mother starter and 0.45 kg of Streptococcus thermophilus mother starter were added to 30 kg of sterilized skim milk with a non-fat milk solid content of 10%, and mixed.
Culture was carried out at ℃. The luminescence amount of the culture solution was measured every 20 minutes during the culture using the same method as in the test example, and the value (ratio) was calculated by dividing the logarithm of the current luminescence amount by the logarithm of the luminescence amount 20 minutes before. After 3 hours and 40 minutes of culture, the ratio reached 1.03, so culture was stopped and about 30 kg of highly active symbiotic starter was collected.
I got it. Example 3 Commercially available Lactobacillus bulgaricus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium, sterile skim milk was inoculated with 3% of the above culture, and incubated at 37°C for 16
After culturing for hours, a mother starter of Lactobacillus bulgaricus was prepared. Separately commercially available Streptococcus
Streptococcus thermophilus (chilled and dried product, manufactured by Hansen) was subcultured three times using sterilized skim milk as a medium, and sterile skim milk was inoculated with 3% of the above culture and cultured at 37°C for 16 hours. A mother starter of Filus was prepared. 0.25 kg of the above Lactobacillus bulgaricus mother starter and 0.25 kg of Streptococcus thermophilus mother starter were added to 30 kg of sterilized skim milk with a non-fat milk solids content of 10%, and mixed.
Culture was carried out at ℃. The luminescence amount of the culture solution was measured every 20 minutes during the culture using the same method as in the test example, and the value (ratio) was calculated by dividing the logarithm of the current luminescence amount by the logarithm of the luminescence amount 20 minutes before. Five hours and 20 minutes after the start of culture, the ratio reached 1.05, so culture was stopped and approximately 30
I got kg.

[0023]

[Effects of the Invention] As explained above in detail, the following effects can be achieved by the present invention. (1) Quickly determine when to stop the culture without measuring the number of bacteria in the culture solution or based on the growth curve of microorganisms.
By making accurate judgments, it is possible to produce a starter with a high bacterial concentration and the highest activity. (2) The time to stop culturing the starter for producing a highly active starter can be determined almost instantly. (3) It is possible to easily determine when to stop culturing the starter to produce a highly active starter.

Claims (1)

[Claims] Claim 1: In the production of a starter, the ATP luminescence amount of the starter culture solution is measured over time at regular intervals, and the ratio of the logarithm of the measured luminescence amount to the logarithm of the previous measured luminescence amount is determined. 1. A method for producing a starter, characterized in that culture is stopped when