Fermented milk and a process for the manufacture thereof

Abstract: Fermented milk with a controlled acidity increase during storage and transportation is disclosed. The fermented milk can be manufactured by the addition to raw milk of nisin-producing lactic acid bacteria belonging to genus Lactococcus lactis subsp. lactis together with other lactic acid bacteria to be used for the fermentation of the raw milk. Nisin produced and accumulated in the fermented milk suppresses the growth of bacteria which cause acid formation, thus controlling the acidity increase during storage and transportation.

Description: The present invention relates to fermented milk exhibiting a controlled increase in acidity during storage and transportation containing lactobacilli and lactococci at a specific ratio, and to a process for manufacturing such fermented milk. Antibacterial substances produced by lactic acid bacteria, typified by nisin produced by Lactococcus lactis subsp. lactis , formerly called Streptococcus lactis subsp. lactis , are used for the purposes of promoting the storage performance of foods and preventing the quality of foods from deteriorating due to proliferation of bacteria in the foods and contamination thereby. It is very difficult to retain through the entire period of storage and transportation favourable flavour possessed by fermented foods immediately after production, e.g., fresh flavour of fermented milk in which living bacteria are present. Yoghurt, for example, which is characterised by the living bacteria therein, possesses an inherent defect in its flavour change due to the increase in acidity during storage and transportation. A number of trials have been undertaken for preventing the increase in acidity of yoghurt during storage. All of them, however, have yielded insufficient results; some having very limited effectiveness in preventing the acidity increase, others required very complicated manufacturing processes, and many of them involved high production costs. As mentioned above, living bacteria contained in fermented food products, such as yoghurt, proliferate during storage and increase the acidity of the products, leading to a deterioration in flavour. Development of a process for preventing such deterioration in flavour in fermented food products by suppressing activities of bacteria, which is a cause of such flavour deterioration, by a simple method without altering conventional process steps has been desired. Amongst bacteria used for fermentation of raw milk at the start of yoghurt production are Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus , the former being the cause of the acidity increase at least in the storage and transportation period. Controlling the growth of Lactobacillus delbrueckii subsp. bulgaricus to directly inhibit the increase in acidity of the product during storage, therefore, leads to efficient prevention of product quality deterioration. Indian Journal of Dairy Science, Vol. 28, No. 1, 71-72, (1975) relates to a process for the preparation of yoghurt by incubating a nisin producing bacteria culture in the yoghurt to inhibit acid production by lactobacillus. The nisin producing bacteria is Streptococcus lactis-4 . The present inventors have undertaken extensive studies on the mutual actions between bacteria used at the start of fermentation (such bacteria are hereinafter called "starter bacteria" or simply "starter") in manufacturing processes of fermented foods, and found that the growth of starter bacteria in the raw milk is suppressed by certain bacteria of Lactococcus lactis subsp. lactis . The inventors have further studied the cause of such inhibition and found that an antibacterial agent nisin secreted by Lactococcus lactis subsp. lactis ATCC 11454 in a medium suppresses the growth of other starter bacteria and controls formation of acids. These findings have led to the the present invention. Accordingly, an object of this invention is to provide fermented milk with a controlled acidity increase during storage and transportation comprising living lactic acid bacteria consisting of a Lactobacillus sp. and a Streptococcus thermophilu sp. and nisin produced by Lactococcus lactis subsp. lactis ATCC 11454. Another object of the present invention is to provide a process for the manufacture of fermented milk with a controlled acidity increase during storage and transportation comprising the addition to raw milk of a nisin-producing lactic acid bacteria belonging to genus Lactococcus lactis subsp. lactis ATCC 11454 together with a Lactobacillus sp. and with a Streptococcus thermophilus sp. and fermenting the raw milk. In a preferred embodiment of the present invention, the lactic acid bacteria comprises Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP- 3762) as Lactobacillus sp., or Streptococcus salivarius subsp. thermophilis as Streptococcus thermophilus sp., or both. Further objects, features and advantages of the present invention are described below. Nisin is a poly-peptide antibacterial compound produced by certain bacteria belonging to genus Lactococcus lactis subsp. lactis , e.g., ATCC11454 strain. Its primary structure is as follows (J. Amer. Chem. Soc., 93 , 4634-4635 (1971). ATCC11454 is listed in the catalogue issued by American Type Culture Collection and can be obtained from the institute. This antibacterial substance has a narrow spectrum and, as described later in more detail, only suppresses the growth of Lactobacilli which are the cause of product quality deterioration and does not suppress the growth of Lactococci. Nisin is produced by the cultivation of nisin-producing bacteria such as Lactococcus lactis subsp. lactis ATCC11454, collected from the culture broth, and purified. Since it is produced by lactic acid bacteria which are used in food fermentation, nisin is a highly safe compound. In the United States, it is added to cheese for the purpose of preventing abnormal fermentation or rot of cheese. Also, for the production of cheese free from abnormal fermentation, nisin-producing bacteria is used as a starter microorganism. According to the present invention, the acidity increase in fermented milk products can be controlled by manufacturing such products using, as starter bacteria, nisin-producing lactic acid bacteria belonging to genus Lactococcus lactis subsp. lactis ATCC 11454. This microorganism can be mutated by conventional mutation treatments, e.g., by irradiation of UV rays or by a treatment with chemicals, e.g., N-methyl-N′-nitro-N-nitrosoguanidine (NTG), etc. The present invention also includes such mutants. Examples of lactic acid bacteria used in the present invention include Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus subst. jugurti , Bifidobacterium breve subst. breve, Streptococcus salivarius subsp. thermophilus , and the like. Among these, Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP- 3762) is particularly preferable as Lactobacillus sp. due to its low acid-characteristic and the high sensitivity toward nisin. The mutants of this bacteria may also be used in the process of the present invention. For carrying out the process of the present invention, these lactic acid bacteria are added to pasteurized milk,such as pasteurized skim milk,and cultivated. Cultured material is used as a starter for the fermentation of milk. The starter may contain both the cultivated nisin-producing bacteria and the lactic acid bacteria for the fermentation, or nisin-producing bacteria and the lactic acid bacteria may be individually cultivated to provide starters. Alternatively, all these bacteria may be directly added to a fermentation raw material. The amount of starter bacteria used for the production of fermented milk according to the present invention is 0.1-5% of the raw milk for nisin-producing bacteria and 1-3% for the lactic acid bacteria. Fermentation can be carried out under the same conditions as those applied to conventional milk fermentation processes. According to the present invention, the growth of lactic acid bacteria for use in the fermentation of milk can be suppressed by nisin produced by these nisin-producing bacteria, thus preventing the quality of fermented products containing these fermentation lactic acid bacteria from deteriorating during storage and transportation. In particular, of the most commonly used starter bacteria, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus , the former is the cause of product quality deterioration during storage and transportation, the growth of which can be suppressed by nisin. Thus, nisin promotes the quality of the fermented milk by retaining the amounts of these two bacteria in a suitable ratio. Fermented milks of the present invention include hard yoghurt, soft yoghurt, acidophilus milk, fermented soft drinks, fermented butter milk, and the like. Other features of the invention will become apparent in the course of the following description of the preferred embodiments which are given for illustration of the invention and are not intended to be limiting thereof. EXAMPLES Example 1 To 2 kg of a yoghurt mix consisting of raw milk, powdered skim milk, and sugar were added 1% of commercially available Streptococcus salivarius subsp. thermophilus and 1% of low acid-producing Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP- 3762 ), followed by fermentation at 42°C for the production of a control sample of fermented milk. Other samples were prepared by adding, in addition to the above two bacteria, 0.1-1.0% of Lactococcus lactis subsp. lactis ATCC11454, followed by fermentation at 42°C. Acidity of each sample was measured over time and the fermentation terminated when the acidity reached 0.65%. Fermented milks thus obtained were stored at 10°C and their acidity measured after 14 days. The results are shown in Table 1, which demonstrates that the addition of ATCC11454 remarkably reduced the increase in acidity, e.g., to 76% of that of the control by the addition of 0.3% of ATCC11454 and to 49% of that of the control by the addition of 1.0% of ATCC11454. This test also indicated that, in the comparison of Sample No. 4 to which ATCC11454 was added together with other lactic acid bacteria at the start of fermentation and Sample No. 5 to which ATCC11454 was added 2.5 hours after the start of the fermentation, the former sample tank a longer time to reach an acidity of 0.65% and its acidity increase was more effectively controlled during the storage. Sample No. ATCC11454 (%) Fermentation time (hour) Acidity (%) Acidity increase in 14 days (%) (8-A) At completion of fermentation (A) After 14 day storage (8) 1 - 3:15 0.642 1.030 0.388 (100%) 2 0.1 3:30 0.647 1.018 0.371 (96%) 3 0.3 3:35 0.651 0.947 0.296 (76%) 4 1.0 6:20 0.637 0.826 0.189 (49%) 5 1.0 5:15 0.647 0.897 0.250 (64%) With the addition of 0.3-1.0% of ATCC11454 the number of living bacteria Lactobacillus delbrueckii subsp. bulgaricus , was controlled to 40-53% of that of the control sample, while the growth of Streptococcus salivarius subsp. thermophilus was hardly affected. Lot No. ATCC11454 (%) cfu/ml 1 - Streptococcus 4.7x10 8 76% Lactobacillus 1.5x10 8 24% 2 0.1 Streptococcus 5.1x10 8 79% Lactobacillus 1.4x10 8 21% 3 0.3 Streptococcus 6.4x10 8 89% Lactobacillus 0.8x10 8 11% 4 1.0 Streptococcus 5.3x10 8 90% Lactobacillus 0.6x10 8 10% 5 1.0 Streptococcus 5.0x10 8 85% Lactobacillus 0.9x10 8 15% Example 2 To 2 kg of a yoghurt mix consisting of raw milk, powdered skim milk, and sugar were added 1.5% of commercially available Streptococcus salivarius subsp. thermophilus and 1.5% of low acid-producing Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP- 3762), followed by fermentation at 42°C with stirring. 3-4 hours after the start of the fermentation, 0-5% of Lactococcus lactis subsp. lactis ATCC11454 was added, and the fermentation was continued at 42°C for 4 hours in total. Fermented milks were then stored at 10°C and their acidity measured after 14 days. The antibacterial activity of ATCC11454 was measured by the 96-well microplate method. Table 3 shows the number of living bacteria (colony forming unit: cfu) in yoghurt when the 4 hour fermentation was completed and changes in acidity during the storage at 10°C. As the table indicates, in the sample to which 5% of ATCC11454 was added, growth of bacteria when ATCC11454 was added 3.0 hours after the start of fermentation was controlled to 43% of that of the control sample (3.0x10 8 /7.0x10 8 ) and growth when ATCC11454 was added 3.5 hours after the start of fermentation was controlled to 61% of that of the control sample (4.9x10 8 /8.0x10 8 ). The acidity increases in these samples were 62% and 81% respectively of that of the control sample, to which no ATCC11454 was added. In the sample to which ATCC11454 was added immediately after the 4 hour fermentation, the growth of bacteria was controlled to 80% (6.7x10 8 /8.4x10 8 ) of that of the control sample and the acidity increase was 83% of that of the control sample. The antibacterial activity of ATCC11454 bacteria on Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 was 12.5 U/ml converted to nisin. ATCC11454 Addition Time: hour Added ATCC11454 (%) Number of bacteria Upon completion of fermentation (cfu/ml) Acidity increase After 7 days After 14 days 3.0 0 7.0x10 8 0.276 (100%) 0.331 (100%) 1 5.7x10 8 0.235 (85%) 0.282 (85%) 2 4.9x10 8 0.230 (83%) 0.278 (84%) 5 3.0x10 8 0.142 (51%) 0.206 (62%) 3.5 0 8.0x10 8 0.287 (100%) 0.315 (100%) 1 7.0x10 8 0.276 (96%) 0.323 (103%) 2 7.4x10 8 0.245 (85%) 0.270 (86%) 5 4.9x10 8 0.203 (71%) 0.256 (81%) 4.0 0 8.4x10 8 0.282 (100%) 0.324 (100%) 1 8.6x10 8 0.285 (101%) 0.326 (101%) 2 6.7x10 8 0.263 (93%) 0.314 (97%) 5 6.7x10 8 0.235 (83%) 0.269 (83%) Example 3 ATCC11454 bacteria were added into Elliker medium with the composition shown in Table 4, in an amount constituting 1% of the medium, and fermented at 30°C for 20 hours. Cells were separated by centrifugation at 5,000 rpm for 15 minutes, washed with physiological saline, and dispersed into an equivalent amount of 12% reduced skim milk. Yoghurt mixes were prepared by adding 1% of commercially available Streptococcus salivarius subsp. thermophilus and 1.5% of low acid-producing Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP- 3762), and the further addition of ATCC11454 bacteria in amounts of 0-5% as listed in Table 5. The mixes were fermented by allowing them to stand at 42°C for 4 hours and stored at 0°C. After 14 days, the acidity of the yoghurt mixes was measured. The antibacterial activity of ATCC11454 upon completion of fermentation was measured by the 96-well microplate method. The results are shown in Table 5. Bacto Tryptone 20 g/l Bacto Yeast Extract 5 Bacto Gelatin 2.5 Bacto Dextrose 5 Bacto Lactose 5 Bacto Saccharose 5 Sodium Chloride 4 Sodium Acetate 1.5 Ascorbic Acid 0.5 ATCC11454 (%) Number of living bacteria (cfu/ml) Acidity (%) Acidity increase (%) Antibacterial Activity (U/ml) Streptococcus Lactobacillus Upon fermentation After 14 days 0 6.7x10 8 3.1x10 8 0.888 1.328 0.443 (100%) 0 0.5 6.3x10 8 2.3x10 8 0.850 1.255 0.404 (91%) 0 1 6.5x10 8 2.2x10 8 0.838 1.239 0.401 (91%) 0 2 6.5x10 8 1.4x10 8 0.800 1.129 0.329 (74%) 2 5 6.5x10 8 0.4x10 8 0.600 0.893 0.291 (66%) 2 As shown in table 5, in the sample to which 5% of ATCC11454 was added the number of cells of Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 was controlled to 13% (0.4x10 8 /3.1x10 8 ) of that in the control during the 4 hour fermentation and the acidity increase was 66% of that of the control. The antibacterial activity of the yoghurt at the completion of the fermentation against Bacillus stearothermophilus was 2 U/ml. Here, the activity to suppress the growth of 10 6 cells of Bacillus stearothermophilus is defined as 1 U/ml of the antibacterial activity. As illustrated above, according to the present invention raw milk is fermented with the addition of a nisin-producing lactic acid bacteria belonging to genus Lactococcus lactis subsp. lactis ATCC 11454 together with starter bacteria, thus ensuring the production of nisin in the fermented milk. The nisin can effectively suppress the growth of lactic acid bacteria, especially of lactobaccili. As a result, the proportion of lactobaccili and lactococci in the fermented milk is kept at a suitable ratio so as to suppress acid production during storage and transportation. The fermented milk retains its quality without being deteriorated by the action of acids and the like.

Claims: Fermented milk having a controlled acidity increase during storage and transportation comprising living lactic acid bacteria consisting of Lactobacillus sp . and a Streptococcus thermophilus sp . and nisin produced by Lactococcus lactis subsp. lactis ATCC 11454, wherein of the bacteria present after fermentation the amount of Streptococcus thermophilus bacteria is 82-94%. Fermented milk according to Claim 1, wherein said living lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP-3762), or Streptococcus salivarius subsp. thermophilus , or both. A process for the manufacture of fermented milk with a controlled acidity increase during storage and transportation comprising: the addition to raw milk of 0.3 to 5% nisin-producing lactic acid bacteria belonging to genus Lactococcus lactis subsp. lactis ATCC11454 together with a Lactobacillus sp . and with a Streptococcus thermophilus sp . and fermenting the raw milk, wherein of the bacteria present after fermentation the amount of Streptococcus thermophilus bacteria is 82-94%. The process according to Claim 3, wherein said living lactic acid bacteria are Lactobacillus delbrueckii subsp. bulgaricus SBT 1511 (FERM BP-3762), or Streptococcus salivarius subsp. thermophilus , or both.

Inventors: AHIKO KENKICHI, ISHII SATOSHI, TOYODA SHUJI, YAMAUCHI YOSHIHIKO, AHIKO, KENKICHI, ISHII, SATOSHI, TOYODA, SHUJI, YAMAUCHI, YOSHIHIKO, ISHII, SATOSHI, KITA 8-JOH HEIGHTS 5,, TOYODA, SHUJI, 1-3, HIROBACHO 1-CHOME, YAMAUCHI, YOSHIHIKO, ELIZABEITO SHINSAPPORO 305
Applicants: SNOW BRAND MILK PROD CO LTD, SNOW BRAND MILK PRODUCTS CO., LTD.
Application year: 1992
Main classification: A23C9
IPC codes: A23C9