GB2251864A

GB2251864A - Lactobacillus strains stable at low temperature

Info

Publication number: GB2251864A
Application number: GB9100915A
Authority: GB
Inventors: Yuan Kun Lee; Siew Fai Wong
Original assignee: MALAYSIA DAIRY IND Pte Ltd
Current assignee: MALAYSIA DAIRY IND Pte Ltd
Priority date: 1991-01-16
Filing date: 1991-01-16
Publication date: 1992-07-22
Anticipated expiration: 2011-01-16
Also published as: GB2251864B; GB9100915D0; MY108214A

Abstract

Lactic acid tolerant, lactic acid producing strains of Lactobacillus are viable for at least 30 days at 5 DEG C. Preformed strains are L. Acidophilus, L.plantarum and L. casei. The said strains may be obtained by culture of a mixed population of Lactobacillus strains in a medium comprising milk at a pH of from 3.4 to 4.2, with addition of fresh medium at a pH below 3.8.

Description

Products and Process The present invention relates to Lactobacillus variants having long shelf-life at low temperature and methods for obtaining them.

The major requirements of a high quality Lactobacillus milk culture are the fermentation of lactose in milk to lactic acid, and a high viable count of the lactic acid bacteria. Sustaining a viable cell count in bottled milk products has become more critical in recent years with emphasis on the possible therapeutic benefits that result from the interaction between lactic acid bacteria and man. There is considerable evidence that replenishment of the intestinal lactobacilli is beneficial.

Several species of Lactobacillus can be isolated from the human intestinal tract but only Lactobacillus acidophilus is present in sufficient numbers to be important. The L. acidophilus, however, has a relatively short stationary phase in its growth cycle, resulting in short shelf-life in fermented milk products in cold storage. The viable cell count could drop more than 4 log values after 2 weeks of cold storage.

In our earlier attempts to isolate mutants of Lactobacillus acidonhilus with long shelf-life by repeated sub-culturing of viable cells in month-long Lactobacillus milk cultures maintained at 5"C, some Lactobacillus strains of long shelf-life were obtained.

However, their long shelf-life in each case turned out to be due to their inability to produce lactic acid, thus rendering them unsuitable for milk fermentation purpose. As the viability of all Lactobacillus cells, in general, could be maintained for extended periods at neutral pH rather better than at low pH, selection by repeated sub-culturing of month-long Lactobacillus cultures without pH control ultimately selects non-acid producing strains.

We have now established that the main factor affecting the viability of Lactobacillus cells during cold storage is the acidity of the fermented milk. The present invention is based on the concept of selecting variant strains of lactic acid producing Lactobacillus strains which are more acid tolerant than previous known strains.

According to the present invention we provide lactic acid tolerant, lactic acid producing strains of Lactobacillus which remain viable for at least 30 and preferably at least 40 days in fermented milk at 5"C.

Such strains are preferably strains of L. acidophilus.

Other possible strains include L. plantarum and L. casei.

The pH of a fermented milk culture rapidly falls from neutral to about 3.6 to 4.0, usually 3.8, and this is the pH value to which the novel strains will be subjected during cold storage. It has been observed that the stationary phase of the novel strains under such conditions is significantly lengthened and the invention also provides novel strains of Lactobacillus, especially L. acidophilus, wherein the stationary phase at 5"C in fermented milk is of at least 30 days and preferably at least 40 days duration.

Novel strains of Lactobacillus may be obtained by mutation of natural strains, for example by mutagenesis, e.g. using radiation such as ultraviolet light, chemical mutagens or recombinant DNA technology, or by somatic hybridisation, i.e. protoplast fusion. The mixed population of strains so produced may then be subjected to the novel selection procedure described below to isolate strains having improved acid tolerance. In this way, we have produced three novel strains of L.

acidophilus, namely MDI1, MDI2 and MDI3, and also a novel strain of L. plantarum, MDI1, and of L. casei, MDI1.

The profiles of these new strains have been studied using the API-50 CH Lactobacillus identification kit and the carbohydrate fermentation profiles are shown in Tables A, B, C and D, which refer respectively to L.

acidophilus MDI1, MDI2, L. plantarum MDI1 and L. casei MDI1. These strains have been deposited at the National Collections of Industrial and Marine Bacteria Limited on 4 January 1991 under the accession numbers NCIMB 40354, 40355, 40356 and 40357 respectively.

The invention also extends to fermented milk containing one or more of the Lactobacillus strains according to the invention.

According to a further aspect of the present invention, we provide a method of selecting a lactic acid producing, lactic acid tolerant Lactobacillus strain viable for at least 30 days in fermented milk at 5"C, wherein an initial mixed population of Lactobacillus strains is cultured in a milk medium at a pH in the range 3.4 to 4.2, fresh medium being added to the culture at intervals or continuously only when the pH is below 3.8, medium and cells being removed from the culture continuously or at intervals to maintain the culture volume within prescribed limits, whereby the medium is enriched in said lactic acid producing acid tolerant strain.

In such a system, the lactic acid concentration at the end of the culture period and at periods at which the pH value is about 3.4, will generally be in the range 0.8 to 1.2% by weight. The culture period for such selection is preferably at least 20 days, more preferably at least 30 days. The fresh medium will normally be unfermented milk which is commonly at about pH 6.2 to 6.5 after autoclaving at 121"C for 15 minutes.

It is believed that the Lactobacillus cells are primarily susceptible to low pH and energy is required Table A Carbohydrate Fermentation Profile of Lactobacillus acidophilus MDI 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 24 48 72 96 Table B Carbohydrate Fermentation Profile of Lactobacillus acidophilus MDI 2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 24 48 72 96 Table C Carbohydrate Fermentation Profile of Lactobacillus Plantarum MDI1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 3 6 24 49 Table D Carbohydrate Fermentation Profile of Lactobacillus casei MDI 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 3 6 24 48 to maintain the intracellular pH values.Variants of Lactobacillus that do not require or require less energy to-maintain their viability at low pH values will have larger portion of energy available for growth, and thus out-grow less acid tolerant cells. These faster growing acid-tolerant variants of Lactobacillus will also survive longer at low pH in stationary phase cultures, so they require less energy to work against the low pH condition in culture medium.

Selection by the process of the invention is automatic. Since medium is continually removed, more slowly propagating cells will eventually be washed out to leave only the faster propagating cells. By adding fresh medium only when the pH is below 3.8, the selective more rapid propagation of acid tolerant strains is ensured. The fresh medium will normally be substantially neutral and will raise the pH, whereafter production of lactic acid by the cells will eventually cause the pH to fall below 3.8. In the event that acid tolerant strains are present which do not themselves produce lactic acid, but nevertheless are propagated, the rate of fall of pH after addition of fresh medium will be relatively slow and thus delay addition of further medium. The various sub-populations of cells will thus compete for the limited supply of medium.It is physiologically unlikely that the acid-producing and non acid-producing cells have the same maximum specific growth rate and affinity for the substrate. If the desired acid-producing cells grow faster, they will rapidly predominate in such a system and become selected. If the undesired non acid-producing cells grow faster, they may predominate so that lactic acid production and hence addition of fresh medium will eventually cease. After a time, such a culture may be rejected unless any sub-population of faster growing acid producing cells has had time to grow into a predominant population and re-start the procedure.

The following example is given by way of illustration only: Lactobacillus acidophilus strains were introduced into a culture vessel containing 500 ml of milk culture medium (pH 6.2, containing 4% non-fat milk-solid).

The culture was agitated mechanically. The pH of the culture was monitored by a pH indicator/controller, which activates a peristaltic pump when the pH of the culture dropped below 3.8. The peristaltic pump delivered fresh milk medium into the culture. The decline in pH of the batch culture was due to the fermentative production of lactic acid. Addition of fresh milk restored the pH of the culture to a value slightly above 3.8 due to dilution by a neutral medium.

The volume of the culture in the vessel was controlled by two level-sensing probes, one fixed at the high level of 1.5 L and the other at the low level of 0.5 L. The signal from the high level sensor was amplified by an electronic liquid level controller to activate a harvest pump, which in turn was deactivated by signals from the low level sensor.

The temperature of the culture was maintained at 37or. The decrease in pH due to the increased amount of lactic acid through Lactobacillus growth in the culture system was in turn counteracted by the addition of fresh medium. Thus, the growth of Lactobacillus could be monitored by measuring the cumulative amount of fresh medium added into the culture system assuming that the cell yield of lactic acid is a constant.

As indicated above, the principle of the selection procedure is that as acid tolerant Lactobacillus cells grow, more milk medium is fed into the culture system.

The dilution rate therefore depends on the overall growth rate of the culture system. While cultures grew, the total volume of the culture increased with time. As the culture level comes into contact with the high liquid level sensor inserted in the culture vessel, a harvest pump is switched on. The pump is switched off when the culture level dropped and contacted the low level sensor. Thus another selection cycle is initiated. The cell concentration remained at about 107 cells/ml due to the dilution of culture by fresh medium as cell grew.

When the pH value in the culture drops below a critical value, growth rate of the culture decreases.

Therefore an increase in the growth rate of the culture indicates an improvement in acid tolerance of the Lactobacillus culture. The higher the growth rate, the faster the fresh medium is added into the culture, and thus the higher the dilution rate. The selection process is therefore fully automatic. The frequency and pressure of selection is therefore determined by the intrinsic potential of the culture. These factors are important in the selection of a small population of superior mutants.

Claims

1. Lactic acid tolerant, lactic acid producing strains of Lactobacillus which remain viable for at least 30 days in fermented milk at 5"C.

2. Lactic acid tolerant, lactic acid producing strains of Lactobacillus wherein the stationary growth phase in fermented milk at 5"C is at least 30 days.

3. Lactobacillus strains as claimed in claim 1 or claim 2 being of the species L.acidophilus, L.plantarum or L.casei.

4. Lactobacillus strain as claimed in any one of the preceding claims being Lactobacillus acidophilus strain NCIMB 40354.

5. Lactobacillus strain as claimed in any one of claims 1 to 3 being Lactobacillus acidophilus strain NCIMB 40355.

6. Lactobacillus strain as claimed in any one of claims 1 to 3 being Lactobacillus plantarum NCIMB 40356.

7. Lactobacillus strain as claimed in any one of claims 1 to 3 being Lactobacillus casei NCIMB 40357.

8. Fermented milk or a milk derived product containing a Lactobacillus strain as defined in any one of the preceding claims.

9. A method of selecting a lactic acid producing, lactic acid tolerant Lactobacillus strain as defined in claim 1 or claim 2 wherein.an initial mixed population of Lactobacillus strains is cultured in a milk medium at a pH in the range 3.4 to 4.2, fresh medium being added to the culture at intervals or continuously only when the pH is below 3.8, medium and cells being removed from the culture continuously or at intervals to maintain the culture volume within prescribed limits, whereby the medium is enriched in said lactic acid producing acid tolerant strain.

10. A method as claimed in claim 9 wherein the culture volume is maintained between 0.5V and 1.5V, wherein V is the average volume of the culture.

11. A method as claimed in claim 9 or claim 10 wherein the culture period for selection is at least 20 days.