JP2598689B2 - Lactic acid bacteria for malolactic fermentation - Google Patents

Description

The present invention relates to a malo-lactic fermentation (Malo-lactic Ferme).
lactic acid bacteria suitable for use in ntation (hereinafter abbreviated as MLF).

2. Description of the Related Art MLF is a fermentation phenomenon that is very important in wine flavor formation, such as decomposing malic acid in wine into lactic acid bacteria by lactic acid bacteria to reduce acidity and increasing aroma components such as ethyl lactate.

By the way, conventionally, it has been usual that MLF is attached to grape fruit or waits for MLF to occur naturally by a wild lactic acid bacterium resident in a brewery. However, MLF caused by such wild lactic acid bacteria does not always occur, and there is a problem that the quality of the finished wine is not constant if left to natural fermentation.

Under these circumstances, recently, studies have been conducted to artificially inoculate mash with lactic acid bacteria to induce MLF, and dry powdered lactic acid bacteria for MLF have already been marketed in Europe and the United States.
However, when examining this commercial powdered lactic acid bacterium,
I learned the following problems.

In other words, the growth of the domestic grape Muscat Berry A in the mash is slow, and the flavor of the finished wine is not very good. For example, when this powdered lactic acid bacterium was used, it took about one month after the completion of alcohol fermentation to complete MLF (although L-malic acid was not detected). This slow growth (slow progression of MLF) is a very serious problem, especially when there is a production time limit on the sales schedule, and the practical value of such strains is low.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned current situation of lactic acid bacteria used for MLF. Another object of the present invention is to provide a lactic acid bacterium for MLF that can impart good flavor to wine.

 Hereinafter, the present invention will be described in detail.

Means for Solving the Problems The lactic acid bacterium according to the present invention can be isolated from wild strains, and more specifically, causes MLF of Muscat berry A species produced by Snow Brand Belforet. Leuconostock (Leuconosto)
c) SBT 0568 belonging to the genus. By the way, this strain SBT
No. 0568 has been deposited under Microfabrication Laboratory No. 10080 (FERM-P No. 10080).

The characteristics of the microbiological properties of the above strain SBT 0568 (FERM-P No. 10080, hereinafter referred to as "strain A") were characterized by a typical European lactic acid bacterium for dry powdered wine (leuconostoc oenos "Leuconostoc oenos" or lower). B) and MLF lactobacillus ATC previously isolated from wine in Japan
C 27307 (Leuconostoc mesenteroides subsp.
subsp.lactosum ", hereinafter referred to as strain C).

Effect of pH Muscat Berry A juice (Sugar content 14%, pH 3.6, L-
Malic acid 6g /. In the following text, juice refers to this juice unless otherwise specified. ) PH with 10% tartaric acid solution
Adjusted to 3.3 and 3.0. Strain A, Strain B and Strain C were cultured in medium 291 at 25 ° C for 72 hours, and then collected by centrifugation.
Approximately 1 × 10 ⁷ / ml was inoculated into the fruit juice adjusted for H and cultured at 25 ° C. for 7 days. The results are shown in FIGS. 1, 2, 3 and 4 (viable cell count, L-malic acid concentration).

(Viable count) In the case of pH 3.3, strain A grew in 24 hours to reach 10 ⁸ . After that, the phenomenon continued, but it continued to maintain 10 ⁷ units. Strain B reached gradually 10 ⁸ units in to 4 days after the growth after inoculation, was then also slightly growth. After the strain C grown to about 10 ^eight 24 hours after inoculation, but decreases once, after one week and again later after two days growth reached nearly 10 ^nine.

For pH 3.0, strains A and C are to grow to 10 ⁸ / ml after 24 hours, at 48 hours after, strain C has been at 10 ^seven, gradually decreases in strain A, after 4 days It remained at 10 ^{to six.} Strain B 10 ^five after 48 hours, lowered to 10 ^two to gradually decrease to 7 days after the later. From the above, the resistance in the low pH range near 3.0 was in the order of C>A> B.

(MLF ability) In the case of pH 3.3, disappearance of L-malic acid, that is, MLF was achieved in all three strains. The time from inoculation to the end of MLF is 72 hours for strain A, about 6-7 days for strains B and C,
The time required for B and C was 1/2 or less, and the MLF ability was excellent.

In the case of pH 3.0, strains A, B, and C are all MLF in one week of culture.
However, strain A had a higher degree of progression of MLF than the other two strains, and one week after inoculation, the concentration of L-malic acid was about 1/2 that of strains B and C.

Furthermore, the production amount of acetic acid was pH 3.3, strain A was 0.2 mg 7 days after inoculation, 0.4 mg / week 2 weeks, whereas strain B was 0.2 mg.
4 and 1.3, strain C was 0.4 and 1.3, and strain A was 1/2 of B and C.
In the following, strain A is also excellent in this respect.

Influence of alcohol Muscat berry A juice was adjusted to a concentration of 8.0 and 12.0% with 99.5% ethyl alcohol, and strains A, B and C were inoculated to about 1 × 10 ⁷ / ml, and then inoculated at 25 ° C. Cultured for a week. The juice was sterilized at 90 ° C. for 10 minutes before inoculation. Changes in the number of viable bacteria after inoculation are shown in FIGS.

At a concentration of 8%, there is no significant difference among the three strains. Strain B is inoculated
After 48 hours, strain A and C were grown in 10 ^eight respectively after 96 hours.

At a concentration of 12%, strain C is severely impaired in growth and inoculation 1
After a week, the viable count fell to 10 ⁷ . On the other hand, strains A and B were almost unaffected and maintained the order at the time of inoculation, and strain A tended to increase slightly. From the above, the strength of alcohol resistance was in the order of strains A>B> C.

Effect of SO ₂ Muscat berry A juice is sterilized at 90 ° C. for 10 minutes, strains A, B and C are inoculated in about 10 ^8, and cultured at 25 ° C. for 24 hours, and potassium metabisulfite (K ₂ SO ₅₎ ) At concentrations of 20 and 50p
pm. This was cultured at 25 ° C. for 8 days, and the number of viable bacteria was measured. The results are shown in FIGS.

At 20 ppm, the growth is hardly affected, although there is some difference between the three strains. Strain C decreases to 10 ⁷ after addition, but 4
10 ^eight after day, after 8 days to grow to 10 to ^nine. Strain B does not decrease the viable cell count by addition, but grows slightly. Strain A
Also to maintain the 10 ^eight not reduced.

At 50 ppm, growth of all three strains is inhibited. Most affected strains C, 10 ⁴ units 48 hours after the addition, 1 to 96 hours
0 after the fall to ^three, but 8 days, to recover to 10 ^four. Two days after the addition, strains A and B decrease to 10 ⁴ and 10 ⁵ , respectively, and 4 days later, strain B also decreases to 10 ⁴ , but after that, the order is maintained or gradually increased and does not die. Reduced to 10 ^three, but strains A and B were reduced to 10 ⁴ units. This is considered to be because the SO ₂ concentration decreases with time. In any case, the SO ₂ resistance of the strain A of the present invention cannot be said to be inferior to other strains.

Influence of culture temperature Sterilized Muscat berry A juice was inoculated with strains A, B and C at about 10 ⁵ / ml and cultured at 18 and 28 ° C for 9 days. The results are shown in FIG. 9 and FIG.

Although the number of viable bacteria varies in the initial, ML
The order of 10 ⁷ progressing F is reached at 18 ° C 4 days after inoculation with strains A and C and 6 days after inoculation with strain B at 28 ° C.
2 days for strains A and C and 2.5 days for strain B. In this temperature range, the growth rate differing by 10 ° C is 2
Double or 1/2. Generally, the temperature at which MLF is performed is 20
It is close to ° C. From the test results, it was found that the growth of the strain A of the present invention at this temperature was equal to or higher than that of other strains.

Effect of sugar concentration (osmotic pressure)
Adjusted to 0% (w / w) and 20% (w / w), p-d with dl-tartaric acid
H was set to 4.5. Strain A, B and C are added to this medium at about 10 ⁷ / ml
The cells were inoculated and cultured at 25 ° C for 7 days. The results are shown in FIGS.

At 10% concentration, each strain grew strongly. Especially strain B exhibited vigorous growth as reach 10 ^nine after 24 hours. 48 hours after the strains C, five days after any strain A reached 10 ^nine, it has been made all the growth good.

At 20% concentration, the effect on growth was different. The number reached 10 ⁹ after 3 days for strain B and 5 days for strain C. But not able to grow up to 10 ⁹ units in strains A, was One Toma to 10 ^eight. That is, the strength of the osmotic pressure resistance was C>B> A. It should be noted that in the actual brewing, MLF is hardly caused in a state where the sugar concentration is high, so that it can be said that osmotic pressure resistance is not a very important factor.

Influence of Cu ^{++ For} the raw grape, bordeaux solution of pesticide (CuSO ₄・ Ca
(OH) ₂ ] (only a very small amount remains in the final wine). It is thought that Cu dissolved in fruit mash together with squeezed juice affects the growth of MLF bacteria. First, Cu ⁺⁺ concentration in the medium 291 is 5 and 10mg / so as CuSO ₄ · 5H ₂ O was added, pH was adjusted to 4.5 with dl- tartrate. After sterilizing this at 115 ° C for 10 minutes, strains A, B and C were inoculated at about 10 ⁷ / ml and cultured at 25 ° C. Figure 13 shows the results.
And 14.

At 5 mg / g, growth inhibition was not observed in all three strains. On the other hand, at 10 mg /, growth inhibition was observed to some extent, and the viable cell count decreased immediately after inoculation in all three strains. However, strains B and C started to grow 2 days after inoculation, and strain A started to grow 3 days after inoculation. In particular, strains B and C were active for subsequent growth and
g / hour reached 10 ⁹ units. Strain A grew more slowly than the former, and was one order lower than 5 mg / hr 7 days after inoculation. Therefore, the growth of the strain A of the present invention is slightly larger than that of the other two strains by Cu. This is also the result of a medium with a high pH (pH 4.5).
It is thought that the effect is even greater for fruit juices with a low content.

Influence of coexisting yeast Muscat berry A juice sterilized at 90 ℃ for 10 minutes,
Strain A, B and C were inoculated at about 10 ⁴ / ml, and immediately thereafter, about 5 × 10 ⁶ / ml of wine yeast W-3 or OC-2 were inoculated.
C. for 7 days. The results are shown in FIGS.

(If W-3 coexist) strain A is four days after inoculation, 10 ^seven, grown to 10 ^eight after 6 days, the growth was filed uneventful. On the other hand, strain B and C are slow growth, due to the presence of yeast in 10 ^six even after 7 days, growth was suppressed.

(Case where OC-2 coexists) The overall tendency is the same as that of W-3. That is,
Strain A 10 ⁷ units in 4 days after inoculation, were grown to 10 ^eight after 6 days. On the other hand, strains B and C grow slowly, and 6 days after inoculation.
It only grew to 10 ⁶ units. In addition, in 18 days after inoculation, 1
Because it was filed in 0 ⁷ units, divide that had continued to gradually growth (which was filed The same applies to the case of the W-3.).

When yeast coexists, the effect of alcohol can be considered, but since the sugar content of the juice used in this test is 14%, it is considered that the concentration of the alcohol produced from this does not affect the growth of each strain. (See Effects of Alcohol).

Contrary to the trends of lactic acid bacteria (prevalence of yeast), W-3, in the coexistence with OC-2 both strains A, 4 days after which the number of viable bacteria strains A has reached 10 ^seven rapidly viable count The viable cell count of W-3 was 10 / ml or less after 6 days, and that of OC-2 was 10 ³ / ml or less after 7 days.

For strains B and C, both W-3 and OC-2 were less affected than strain A, and the viable cell count again decreased after 4 days, but maintained a viable cell count of 10 ⁴ / ml or more after 7 days. Was.

The characteristics of the strain A (FERM-P No. 10080) according to the present invention described above in comparison with the strains B and C are summarized below.

(1) L-malic acid which is the most important as MLF
The function of converting to lactic acid, that is, MLF ability, is pH 3.3-3.0
In the low pH range, strain A was significantly better than the other two strains.

(2) When MLF was performed in the fruit juice state, the amount of acetic acid produced by strain A was less than 1/2 that of the other two strains. This has good consequences on sake quality.

(3) Strain A has a normal alcohol concentration of 12% in wine
Alcohol resistance in the vicinity was the best among the three strains.

(4) Yeast A can grow regardless of the presence of wine yeast, but the growth of the other two strains was suppressed by the presence of wine yeast.

(5) Strain A has an osmotic pressure resistance of about 20% of sugar and Cu ⁺⁺
Although 10mg / degree of resistance to concentrations is slightly worse than the other two strains, SO ₂ (concentration 50 ppm) resistance, temperature 18 ° C. and 28 ℃
Growth was equal or higher.

Hereinafter, results of performing MLF using the strain A according to the present invention will be shown as examples.

Example Muscat Berry A juice (conversion sugar 14%, L-malic acid 6.8 g /, pH 3.5) to the converted potassium metabisulfite such as the SO ₂ concentration 50 ppm. This was dispensed into six 500 ml Erlenmeyer flasks by 500 ml at a time and divided into three groups of two. Each group is (a), (b),
Then, the wine yeast OC-2 was inoculated at 5 × 10 ⁶ / ml into all the juices, and the culture was started at 20 ° C. Thereafter, (a), (b) and (c) were inoculated with the bacterial strain A of the present invention and the bacterial strain B for comparison at 10 ⁶ / m, respectively, as follows.
LF was performed.

One day after yeast inoculation.

Two days after sugar supplementation (7 days after yeast inoculation).

After completion of alcohol fermentation (two weeks after yeast inoculation).

 In addition, sugar supplementation was performed up to 22%.

The progress of MLF was shown by the transition of L-malic acid concentration as shown in FIGS.

As can be seen from each figure, strain A almost completely lost L-malic acid within 7 days after inoculation, and MLF was terminated. On the other hand, strain B required more than 30 days.

In the case of the strain A, the MLF was completed about 2 weeks after the inoculation, whereas in the case of the strain B, it took more than one month.

In the case of the strain A, the MLF was completed about 18 days after the inoculation, but in the case of the strain B, the completion of the MLF could no longer be expected (the viable cell count was 1
0 ⁶ / ml or less).

Above, at any of the three inoculation times, when using strain A, MLF is more reliable than strain B,
And it ended much earlier. The quality of the produced wine was also good.

[Brief description of the drawings]

1 to 4 show changes in the viable cell count at each pH when strains A, B and C are inoculated into Muscat berry A juice and cultured. FIGS. 5 and 6 show the changes in the viable cell count after inoculation of strain A, strain B and strain C at each alcohol concentration in the juice of Muscat Berry A, respectively. 7 and 8 diagram shows the strain A at each concentration of SO ₂ in Muscat Berry A juice, the transition of the number of viable bacteria strains B and strains C. Figures 9 and 10 show the effect of culture temperature on Muscat Berry A juice for strains A, B and C. 11 and 12 show the effect of sugar concentration on the culture of strain A, strain B and strain C in a medium based on medium 219. Figures 13 and 14 show the effect of Cu ^{++ on} the growth of strain A, strain B and strain C. FIGS. 15 and 16 show the effect of coexisting yeast on the three strains when Muscat Berry A juice is inoculated with strains A, B and C and cultured. 17 to 19 show the progress of MLF when the above three strains were inoculated into Muscat berry A juice and malolactic fermentation (MLF) was carried out, with the change of L-malic acid concentration.

Claims (1)

(57) [Claims] 1. A lactic acid bacterium SBT0568 for use in malolactic fermentation belonging to the genus Leuconostoc.
(Microtechnical Lab No. 10080).