Fermentation method and compositions including a lactobacillus species strain.

Backqround of the Invention

0001The present invention relates to concentrates of cells of a Lactobacillus having the essential identifying characteristics of Eactobacillus sp. NRRL-B-15,036 which are useful for fermenting foods, particularly meat. The new strain ferments dextrose, but not lactose or sucrose, to produce lactic acid in the food.'

Prior Art

0002The present invention relates to an improvement over the invention in U.S. Patent No. 4,303,679 to Raccach wherein Pedzococcus pentosaceus hSRL-B-11,465 is described for meat fermentations. The present invention also relates to an improvement over U. S. Patent No. 3,814,817 describing Lactobacillus ptantarum NRRL-B-5461 and over U.S. Patent No. 3,561,977 to Rothchild et al describing Pediococcus cerevisiae for meat fermentations. There is a large body of prior art in this field and. these patents. are only representative.

0003LactobaciZZus and Pediococcus cultures are used for meat fermentations with added dextrose to produce lactic acid. These cultures are also able to ferment available sucrose and lactose to produce lactic acid. The problem is that, when uncontrolled quantities of milk solids or other fillers containing lactose and/or sucrose are added to the meat formulation, the cultures can lower the pH to too low a level and spoil the sausage. Thus careful process control of pH is required. Certain European sausages contain significant amounts of lactose and sucrose and can particularly present a problem. This problem is aggravated with Lactobacillus plantarum NRRL-B-5461 and with Pediococcus pentosaceus NRRL-B-11,465, which are commercially available cultures, since they ferment sucrose and lactose rapidly. Another prior art problem is to provide a culture which will ferment in the meat and lower the pH at relatively low temperatures i.e. about 23.9°C (75°F) in a short period of time.

0004Until the present invention, as far as applicant is aware, the prior art has not provided bacterial cultures adapted for meat fermentations which wilL ferment.dextrose and not sucrose and lactose to produce lactic acid and form acid rapidly at relatively low temperatures.

Objects

0005It is therefore an object of the present invention to provide a newly isolated strain of Lactobacillus which does not have the ability to ferment lactose or sucrose and yet retains the ability to ferment dextrose to produce lactic acid in a food. It further is an object of the present invention to provide novel bacterial concentrates which maintain their capability for producing lactic acid from dextrose and will not revert to having the ability to ferment lactose and sucrose as a function of time and repropagation. of the cells. These and other objects will become increasingly apparent from the following description.

General Description

0006The present invention relates to a concentrate containing a substantial proportion of cells of a Lactobacillus having the essential identifying characteristics of Lactobacillus sp. NRRL-B-15,036 which ferments dextrose to produce lactic acid and is unable to ferment lactose or sucrose to produce lactic acid, wherein the concentrate contains at least about 1 x 10⁸ cells per ml in a nutrient medium and is in a storage stable form. The nutrient medium contains a carbohydrate source, essential minerals and a nitrogen source as is known to those skilled in the art. Various known culture preservation agents and techniques can be used to stabilize the bacterial concentrates for storage as is well known"in the prior art. Glycerol is preferred for frozen concentrates as a freezing stabilizing agent. Lyophilized bacterial concentrates can also be prepared as described in U.S. Patent No. 4,205,132. Generally the concentrates contain at least 1 x 10⁸ cells per ml in order to provide a useful number of cells for the fermentation of the food. The cells are used in the food in concentrations between 1 x 10⁵ to 1 x 10⁸ cells per ml or per gram.

0007The present invention further relates to an improvement in the method for preparation of a fermented product by providing a culture in a food containing dextrose and then fermenting the food which comprises: providing a culture which is substantially a Lactobacillus having the essential identifying characteristics of Lactobacillus sp NRRL-B-15,036 which ferments dextrose to produce lactic acid and which does not ferment sucrose or lactose to produce lactic acid in the food and fermenting the food to produce a fermented product. The food is preferably meat.

0008The method and bacterial compositions can include other bacteria used in food fermentations as is known to those skilled in the art. Generally at least a substantial portion (more than about fifty percent by volume) of the concentrate will be LactobaciZZus sp. NRRL-B-15,036. It would be counter-productive to use lactose and/or sucrose fermenting strains in the concentrate.

0009Stimulatory metal salts can be added to the Lactobacillus sp. NRRL-B-15,036 as described in U.S. Patent No. 4,303,679 to Raccach. Manganese ion is preferred.

0010The following is a description of the steps in the isolation of Lactobacillus sp. NRRL-B-15,036, the preparation of bacterial concentrates and then the preparation of sausage. This strain was isolated from sausage containing sucrose and/or lactose fermenting strains as virtually the total flora (83%). It was very unexpected that this strain would be isolated.

Isolation of NRRL-B-15,036

0011Various strains were isolated from sausage on Lactobacilli MRS_TM agar. MRS is a selective medium designed for isolating Lactobacillus type lactic acid bacteria and is available as a powder from Difco, Detroit, Michigan. A broth is prepared by adding water. MRS agar contains about 1.5% by weight of agar added to the broth. Carbohydrates are added to a fermentation broth containing the same ingredients as MRS without dextrose and with bromocresol purple added as pH indicator for determining the carbohydrate fermentation characteristics as described hereinafter. LactobaciZZus sp. NRRL-B-15,036 was picked from the MRS agar and then grown in a 350 ml flask at 35°_C for 24 hours in MRS broth. The medium was then cooled to 4°C and neutralized to a pH of 6.0. The culture was centrifuged and resuspended in about 1/10th the original volume of the culture in spent MRS broth. About 1% by volume of sterile glycerol was added along with about 18.2₅ percent by weight of manganese sulfate monohydrate. The concentrated culture was then rapidly frozen on dry ice. The frozen concentrated culture contained about 83 x 10⁸ cells per ml.

0012The strains were tested for lactose, sucrose and dextrose fermentation using a Mintek® system. The isolated strains including NRRL-B-15,036 were also tested for growth at 15°C and 45° for 64 hours with dextrose (1.0% by weight) and for growth in fermentation broth with the sucrose or lactose (1.0% by weight) using bromocresol purple as an indicator which changes from purple to yellow as the p_H is lowered to about pH 5.2 due to the generation of lactic acid. The results are shown in Table I for NRRL-B-15,036 and for other strains 1 to 5 representing the predominant flora of the sausage. <tables id="tabl0001" num="0001"><img file="EP0104444A2_D0001.tif" /></tables>

0013It was found that even after 33 days, NRRL-B-15,036 would not ferment sucrose or lactose to produce lactic acid in a favorable growth medium. The fermentation pattern identifying characteristics of Lactobacillus sp. NRRL-B-15,036 are shown in Table II in comparison to Lactobacillus coryniformus subspecies torqueus. Strain NRRL-B-15, 036 is similar to this subspecies as can be seen from Table II which is based on Bergy's manual pgs 580 and 586 but it would not be concluded that these strains were the same based upon these test results only. <tables id="tabl0002" num="0002"><img file="EP0104444A2_D0002.tif" /></tables>

0014Lactobacillus coryniformus subsp torqueus has not previously been identified as being useful for food fermentations, particularly meat fermentations and has the essential identifying characteristics of Lactobacillus sp. NRRL-B-15,036 in that it is a Lactobacillus which will not ferment sucrose or lactose to produce lactic acid.

Bacterial Concentrates

0015Bacterial concentrates of NRRL-B-15,036 were prepared as described in U. S. Patent No. 4,303,679 to

Raccach.

0016During growth of strain NRRL-B-15,036 the _pH of medium was maintained at about 6.0 by adding ammonia until the dextrose was exhausted. The cells were concentrated by centrifugation and the resulting pellet was resuspended with supernatant medium to about 1/10 of the original volume. Glycerin was added at a concentration of 10_% (_W/W). Manganese sulfate monohydrate was added in the amount of 18% by weight to some samples and not others. Manganese. ion contributes to the storage stability of the concentrates. The concentrate was frozen for storage and shipment prior to use and was found to be storage stable. The frozen concentrate contained about 14.5 x 10⁹ cells per ml.

Sausage Preparation

0017The following Examples show the preparation of sausage at various temperatures.

Example 1

0018Pepperoni sausage was made at 23.9°C (75°F) using Lactobacillus sp. NRRL-3-15,036. The meat formulation was 2724 grams (6 pounds) of a mixture of 70_% pork and 30% beef, coarsely chopped, to which was added: <tables id="tabl0003" num="0003"><img file="EP0104444A2_D0003.tif" /></tables>

0019In .(A) the culture concentrate was added at a level of about 4 x 10⁷ cells per gram of the meat formulation.

0020The inoculated meat mixture was stuffed into fibrous pepperoni casing and incubated at 23.9°C (75°F) internal temperature at 80% relative humidity. The results are shown in Table III. <tables id="tabl0004" num="0004"><img file="EP0104444A2_D0004.tif" /></tables>

0021As can be seen Lactobacillus sp. NRRL-B-15,036 is very effective in producing sausage at low temperatures.

Example 2

0022To determine if manganese ion stimulates Lactobacillus sp. NRRL-B-15,036 and to determine that the manganese ion and/or nutrients in concentrated preparation of Lactobacillus sp. NRRL-B-15,036 did not stimulate normal microbial flora of meat, the meat fermentation of Example 1 was repeated at an internal meat incubation temperature of 23.9°C (75°F) except-as follows: <ul id="ul0001" list-style="none"><li>In (A) the culture concentrate was added at a level of about 4 x 10⁷ cells per gram of meat. In (_B) manganese sulfate monohydrate was added in the amount of 18% by weight to the culture concentrate prior to freezing and the manganese ion was present in an amount of about 4 ppm of the meat formulation. In (C) the culture concentrate was killed by autoclaving 15 min. at 121⁰C and then manganese sulfate monohydrate was added in the same amount as in (_B) and manganese ion was present in an amount of about 4 ppm of the meat formulation. The-results are shown in Table IV.<img file="EP0104444A2_D0005.tif" /></li></ul>

0023As can be seen from Table IV, Lactobacillus sp. NRRL-B-15,036 is significantly stimulated by manganese ion in the meat. As can be seen by column (C), the manganese ion or nutrients in culture concentrate do not stimulate microbial flora normally present in meat.

0024It was noted that the overall speed (acid production) of cultures (A) and (B) was slower than - expected (see Example l above). It was felt that the unusually low starting pH (5.65) of the meat may have been the reason for the slower fermentation, therefore, the experiment was repeated with cultures (A) and (B) on another batch of meat. The results are shown in Table V-a. <tables id="tabl0005" num="0005"><img file="EP0104444A2_D0006.tif" /></tables>

0025As can be seen from results,Lactobacillus sp. NRRL-B-15,036 is significantly stimulated by manganese ion in the meat and the higher initial pH of the meat reduced the fermentation time.

Example 3

0026The meat fermentation of Example 1 was repeated at an internal meat incubation temperature of (32°C) 90°F except that sausage (B) was inoculated with a commercially available culture of Pediococcus pentosaceus NRRL-B-11,465. The results are shown in Table V. <tables id="tabl0006" num="0006"><img file="EP0104444A2_D0007.tif" /></tables>

0027As can be seen from Table V Lactabacillus sp. NRRL-B-15,036 was not as fast as Pediococcus pentosaceus NRRL-B-11₈465 at 32°C.

Example 4

0028The meat fermentation procedure of Example 1 was repeated at an incubation temperature of 20.6°C (70°F) internal meat temperature. The results are shown in Table VI. <tables id="tabl0007" num="0007"><img file="EP0104444A2_D0008.tif" /></tables>

0029As can be seen from Table VI, the NRRL-B-15,036 rapidly ferments dextrose in the meat at low temperatures.

Example 5

0030The procedure of Example 1 was used for making summer sausage with NRRL-B-15,036 alone (A); and compared to Pediococcus pentosaceus NRRL-B 11,465 (B) with the usual level of dextrose added. This was done to make certain there were no objectionable flavors produced by NRRL-B-15,036.

0031The meat formulation was: <tables id="tabl0008" num="0008"><img file="EP0104444A2_D0009.tif" /></tables>in 4540 grams (10 pounds) of ground beef. Cultures were added at a level of about 4 x 10⁷ cells per gram. The sausage was incubated at (32°C) 90°F at 90% relative humidity until a pH of 4.9 to 5.0 was produced. The initial meat pH was 5.80. The results are shown in Table VII. <tables id="tabl0009" num="0009"><img file="EP0104444A2_D0010.tif" /></tables>

0032There was no difference in flavor based upon triangulation taste tests by expert tasters.

0033It was found that the frozen bacterial concentrates were stable to storage over long periods of time (4 months) without significant loss of viability.

Example 6

0034The procedure of Example 1 was repeated except lactose or sucrose was used instead of dextrose and incubation temperature was 27°C (internal) at 80% relative humidity. The results are shown in Table VIII. <tables id="tabl0010" num="0010"><img file="EP0104444A2_D0011.tif" /></tables>

0035Clearly lactose and sucrose are not fermented by the NRRL-B-15,036.