A method for growing acid producing bacteria in the presence of an essentially water insoluble or a temporarily water insolubilized and thus initially solid form of a neutralizing agent in a growth medium is described. The water insoluble or insolubilized neutralizing agent is a base, basic salt or mixture thereof adapted to provide a controlled reaction with the acid produced by the bacteria without substantially raising the ph of the growth medium. Preferably the neutralizing agent is in a water insoluble form. bulk starter compositions for growing the bacteria including the insoluble or the insolubilized neutralizing agent are also described. Further, bacterial compositions with enchanced storability and viability because of the insoluble or the insolubilized neutralizing agent are described. The method and bulk starter compositions are particularly adapted to growing lactic acid producing bacteria which are then used in making food and beverage products for animals and humans.
|
8. An acid producing bacterial composition for use in a food fermentation which comprises in admixture:
(a) acid producing bacteria which have been grown in an aqueous growth medium including an assimilable carbohydrate source and a nitrogen source to a concentration of at least about 105 cells per ml; and (b) an essentially water insoluble non-toxic basic neutralizing agent such that when acid is produced by the bacteria at least a part is neutralized, wherein the neutralizing agent is a magnesium phosphate or magnesium ammonium phosphate, and phage inhibitory ingredients in an amount sufficient with other media ingredients to inhibit lactic bacteriophage, wherein the neutralizing agent is present in an amount between about 0.1 and 10 parts by weight of the agent per 100 parts by weight of the cells and wherein the amount of the neutralizing agent with the bacteria provides sufficient neutralizing agent in an aqueous growth medium initially to which the cells are to be introduced and grown to maintain the ph above about 5 with the medium and cells in contact with the solid medium over a period of time such that without the neutralizing agent the ph would be reduced to below about ph 5, wherein the growth medium and neutralizing agent are adapted for growing the acid producing bacteria to be used in fermenting the food.
1. In a method for growing acid producing bacteria to be used in fermenting foods by inoculating the bacteria into a growth medium containing water and nutrients for the bacteria and then growing the bacteria in the growth medium and using the bacteria so grown for fermenting the food the improvement which comprises:
(a) providing in the growth medium an essentially water insoluble non-toxic basic neutralizing agent in the growth medium such that a portion of the neutralizing agent remains in solid form in the medium, wherein the neutralizing agent is a magnesium phosphate or magnesium ammonium phosphate , and phage inhibitory ingredients in an amount sufficient with other media ingredients to inhibit lactic bacteriophage, wherein an amount of the neutralizing agent is added initially to the growth medium prior to the generation of acid by the bacteria which is at least sufficient to maintain the ph above about 5 over a period of time such that without the neutralizing agent the ph would be reduced to below about ph 5 and wherein the growth medium and neutralizing agent are adapted for growing the acid producing bacteria to be used in fermenting the food; (b) growing the bacteria in the growth medium in the presence of the neutralizing agent with the medium and cells in contact with the neutralizing agent, wherein a ph range in the medium is maintained so as to promote growth of the bacteria by a controlled reaction of the neutralizing agent with at least a part of the acid produced by the bacteria in the medium over a period of time; and (c) fermenting the food with the bacteria after they have been grown in the growth medium including the neutralizing agent.
6. A bulk starter medium for growing acid producing bacteria for use in a food fermentation comprising in admixture:
(a) a powdered or aqueous bacterial growth medium including a carbohydrate source and a nitrogen source assimilable by the bacteria wherein the powder can be dissolved or dispersed in water to provide the aqueous growth medium with an initial ph between about 4 and 8.5; and (b) an essentially water insoluble non-toxic basic neutralizing agent, in an amount which produces between about 0.1 and 10 parts by weight of the neutralizing agent per 100 parts of the aqueous growth medium, wherein the neutralizing agent is a magnesium phosphate or magnesium ammonium phosphate , and phage inhibitory ingredients in an amount sufficient with other media ingredients to inhibit lactic bacteriophage, wherein a portion of the neutralizing agent remains in solid form in the aqueous medium over a period of time, wherein the amount of neutralizing agent in the bulk starter provides sufficient agent in the aqueous growth medium initially to which the cells are to be introduced and grown to maintain the ph above about 5 with the medium and cells in contact with the neutralizing agent over a period of time such that without the neutralizing agent the ph would be reduced to below 5 and wherein the neutralizing agent maintains a ph in the aqueous medium at a level which provides for growth of the bacteria by a controlled reaction of the neutralizing agent with at least a part of the acid produced by the bacteria over a period of time in which they are grown, wherein the growth medium and neutralizing agent are adapted for growing the acid producing bacteria for use in the food fermentation.
2. The method of
4. The method of
5. The method of
7. The bulk starter of
9. The bacterial composition of
10. The bacterial composition of
11. The bacterial composition of
13. The composition of
14. The composition of
16. The method of
18. The method of
19. The starter medium of
21. The starter medium of
22. The composition of
24. The composition of
|
|||||||||||||||||||||||||||||||||||||||||
1. Field of the Invention
The present invention relates to a method and to bulk starter compositions for growing acid producing bacteria by using an essentially water insoluble or temporarily water insolubilized and thus initially solid form of a neutralizing agent in the growth medium which is adapted to provide a controlled reaction with the acid produced by the bacteria without substantially raising the pH of the growth medium. In particular, the present invention relates to a preferred method wherein the solid form of the neutralizing agent maintains a selected pH range in the growth medium by a controlled reaction of an insoluble form of the neutralizing agent over a period of time with the acid produced by the bacteria.
2. Prior Art
Encapsulating techniques and encapsulated products for the controlled release of materials as a function of the destruction of the encapsulating agent over a period of time are well known to the prior art. For instance, Gutcho, M. H., 1976. Microcapsules and Microencapsulation Techniques. Noyes Data Corp. Park Ridge, N.J. provides many examples of both.
In the field of microbiology, time-releasing capsules have been used to provide for the delayed controlled release of components to bacteriological media where identifying test reactions of the released components are required (Sveum, W. H. and P. A. Hartman, 1977. Appl. and Environ. Microbiol. 33:630-634; Lanz, W. W. and P. H. Hartman, 1976 Appl. and Environ. Microbiol. 32:716-722). They also have been used for the delayed release of nutrients in growing mushrooms (Carroll, A. D. and L. C. Schisler, 1976. Appl. and Environ. Microbiol. 31:499-593); for the delayed release of gluconic acid as an acidulating agent during the smoking of sausage (Rugala, W., 1978. Food Engineering 50:174); and, for incorporating ripening agents into cheese (Magee, E., Jr. and N. F. Olson, 1978. Proceedings 73rd Annual Meeting Am. Dairy Sci. Assn., p. 114).
In prior art unrelated to microbiology, methods and compositions for controlling pH by use of encapsulated alkaline or acid releasing materials are described for instance in U.S. Pat. No. transition metal salts such as manganese and magnesium salts. Many variations in growth media are described in the prior art and will be obvious to one skilled in the art.
The bulk starters include the insoluble or insolubilized solid neutralizing agent in a powdered or aqueous growth media with the growth materials. The media generally have a pH in water of between about 4 and 8.5. The solid neutralizing agent maintains a pH in the medium of above about 5 during growth of the bacteria.
The bacterial compositions can be used directly for fermentation or stored for a period of time. They can be concentrated, particularly mechanically as by centrifuging, and frozen or lyophilized for storage using various conventional stabilizing agents. All of these variations are well known to those skilled in the art.
It has been found that by incorporating between about 0.1 and 10 parts by weight of the insoluble or insolubilized, solid neutralizing agent per 100 parts of the growth medium by weight containing at least about 1×105 cells per ml that the compositions are more stable upon storage. Preferably the bacteria are grown to a concentration above about 1×108 cells per ml, or even 1×109 cells per ml, and then mechanically concentrated from above 1×109 cells per ml up to about 1×1015 cells per ml. It is contemplated that the cells could be grown in a conventional manner, preferably using liquid or gaseous neutralizing agents periodically added to the medium, and then the insoluble or insolubilized neutralizing agent added to provide stability upon use or storage. Between about 0.1 and 10 parts by weight of the insoluble or insolubilized neutralizing agent are added per 100 parts by weight of the concentrated cells.
Examples of the present invention are set forth hereinafter. It is intended that they be only illustrative. The depository for the cultures is Oregon State University in Corvallis, Oregon and the cultures are freely available without cost to the public.
Sodium carbonate as the neutralizing agent was mixed with one percent magnesium stearate as a lubricant and compressed into tablets. Any commercial tablet making machine will work and in this example a model TPK-12t.m. tablet making machine made by Chemical and Pharmaceutical Industry Company was used. The tablets were spray coated in a rotating coating pan with a mixture of by weight 50% methyl cellulose (Methocel® E15) and 50% ethyl cellulose (Ethocel® 45) with a 1%, 2%, 3%, 4%, 5% and 6% coating by weight. The thickness of the coating is dependent on the tablet size and these tablets weighed about 0.310 g. Other tablets sizes and various amounts of coating mixtures were used successfully.
A growth medium was prepared by suspending 86 g of Actilac® a conventional bacterial growth medium including dried whey, nonfat dry milk, sodium citrate and dried autolyzed yeast, in 700 ml of water with stirring at 50 rpm and heating to 80°C Thirty-two (32) tablets containing sodium carbonate with a 5% coating of the cellulose ethers were added and the mixture was maintained at 80°C for 45 minutes. A control growth medium was prepared in an identical manner without the sodium carbonate tablets.
After cooling to 27°C, the medium was inoculated with Strepococcus cremoris 108 at a level of 104 to 106 cells per ml and the pH was monitored in comparison to the control medium without added tablets. The release of the sodium carbonate to the medium is controlled to a certain extent by adjusting media agitation or stirring. The pH immediately after inoculation was 6.29 for the control and 6.41 for the invention; after 14.5 hours, the pH was 4.81 for the control and 5.20 for the invention. In a second determination, the medium containing tablets with a 6% coating had an initial pH of 6.36 compared to 6.32 for the control and a pH after 19 hr. of 5.32 compared to 4.76 for the control. This shows a considerable control of the pH, using a temporarily insolubilized neutralizing salt.
Activity testing by growth in nonfat milk medium for 6 hours at 30° C. using a 1% by volume bacterial inoculum in a 11% by weight nonfat milk medium showed the bacterial composition prepared by addition of sodium carbonate tablets with the 6% coating had the maximum possible activity for the bacteria as determined by comparison to a culture grown conventionally by a continuous addition of ammonium hydroxide for pH maintenance at pH 6∅
One kg of anhydrous granular sodium hydrogen phosphate dibasic was mixed with 50 g of ethyl cellulose (Ethocel® 45) of Example 1. To this mixture, 300 ml of a 70/30 percent by volume mixture of methylene chloride and ethanol 95% was added and mixed for 5 minutes. The mixture was pressed through a number 6 screen to produce granules. The granules were dried, tableted and coated with a 6% coating of methyl cellulose and ethyl cellulose (Methocel® E15/Ethocel® 45) 70%/30% by weight.
The tablets were placed in the growth medium of Example 1, agitated at 64 rpm. The initial pH values were 7.11 for the invention and 6.43 for the control. After 12.25 hours the pH was 6.68 for the invention and 4.92 for the control and after 33.25 hours the invention pH was 5.62 and the control pH was 4.93. In a separate test where the medium containing the tablets was not stirred until about 4.75 hours post inoculation, the initial invention pH was 6.44 and 6.35 for the control and after 16 hours was 5.29 for the invention and 4.81 for the control. In each of the above examples the activity of the bacteria was greater for the media containing the temporarily insolubilized neutralizing salt than for the control.
A growth medium was prepared consisting of a mixture of the following ingredients by weight: 3.5% sweet whey powder; 0.5% yeast extract; 0.5% potassium phosphate dibasic; 0.25% potassium phosphate monobasic. After dissolving the mixture in water, it was autoclaved at 121°C for 10 minutes and then rapidly cooled. To the cooled mixture was added 5 gm of temporarily insolubilized particles which had been prepared by mixing 10 g of carboxypolymethylene (Carbopol 941 ™) with 300 gm of ammonium hydrogen phosphate dibasic and with 50 ml of water, spreading the mixture out to dry at 45°C overnight and breaking the mixture into pieces.
A 1% by volume inoculum of a Streptococcus lactis frozen culture (Fargo® mixed strain starter culture No. 1105) commercially available from Microlift Technics, Inc., Sarasota, Florida containing about 109 cells per ml, was then added to the medium with the insolubilized particles. The fermentation mixture was incubated at 30°C with gentle agitation over a 12 hour period. The pH of the fermentation was continuously recorded by a strip chart recorder. A second fermentation was performed in exactly the same manner, except the insolubilized ammonium phosphate was omitted from the growth medium.
The initial pH of the media was 6.7; after 8 hours the medium of the invention was pH 6.6 and was 5.1 for the control and after 12 hours the pH for the invention was 5.1 and was 4.7 for the control.
During a 10 day storage period of the bacteria at 5°C, measurements of activity in nonfat dry milk were conducted. The medium of the invention produced cells with activity which were superior to the cells produced in medium without the controlled release as shown by Table I.
| TABLE I |
| ______________________________________ |
| Storage Time Activitya |
| (hrs.) Controlled Release Base |
| Control |
| ______________________________________ |
| 0 5.30 5.36 |
| 8 5.25 5.48 |
| 96 5.28 5.94 |
| 240 5.36 6.33 |
| ______________________________________ |
| Final pH after incubation at 30°C for 6 hr. of a 1% by volume |
| inoculum in a 11% by weight solids nonfat milk medium. |
A growth medium was prepared from the following ingredients: 3.5% sweet whey powder; 0.5% yeast extract; 0.5% sodium beta-glycerophosphate; 0.83% sodium citrate; 0.17% ammonium citrate dibasic. After the mixture was dissolved in water, it was heated with high agitation to a temperature of 85°-90°C and held there for 45 minutes. The heat-treated medium was then rapidly cooled in an ice bath. The controlled release ingredient consisted of 10 g of a 50/50 by weight mixture of sodium carbonate tablets coated with 1% or 1.25% by weight of ethyl cellulose (Ethocel® 45). The coating was applied by spraying a solution of ethyl cellulose dissolved in methylene chloride onto tumbling tablets in a standard tablet coating pan.
The medium was then inoculated with Streptococcus lactis 131 B1 (Oregon State University) (1% by volume containing 108 cells per ml) and incubated at 27°C with gentle agitation over a 16 hour period to provide the release of the neutralizing agent. A record of the fermentation pH was made using a strip chart recorder.
A second fermentation was done exactly in the same manner except the controlled release component had a 10% by weight ethyl cellulose coating which prevented adequate release of the sodium carbonate and the pH was not maintained above 5∅
Following the 16 hour fermentation, a portion of the fermentation mixture was stored at 21°C for a period of 10 days during which measurements of starter cell activity and cell numbers were periodically made. The controlled release medium which maintained the pH above 5.5 throughout the 16 hour period resulted in starter cells that had greater activity and higher numbers compared to the fermentation where the pH went below 5.0 where acid injury was evident. The data is presented below for both the pH during the fermentation in Table II and activity during storage in Table III.
| TABLE II |
| ______________________________________ |
| Fermentation Time |
| Controlled |
| (Hours) Release Control |
| ______________________________________ |
| 0 6.7 6.3 |
| 2 6.9 6.4 |
| 4 6.8 6.5 |
| 6 6.2 6.5 |
| 8 5.9 6.0 |
| 10 5.7 5.1 |
| 12 5.6 4.8 |
| 16 5.6 4.7 |
| ______________________________________ |
| TABLE III |
| ______________________________________ |
| Stor- Controlled Release |
| Control |
| age Activity (pH) Activity (pH) |
| Time 6 hr - 6 hr - |
| (Days) |
| 30°C |
| SCCa |
| cfu/mlb |
| 30°C |
| SCCa |
| cfu/mlb |
| ______________________________________ |
| 0 4.76 4.93 8.1 × 109 |
| 4.75 4.95 1.4 × 1010 |
| 1 4.79 4.96 8.8 × 109 |
| 4.84 -- 1.4 × 1010 |
| 2 4.81 4.95 8.2 × 109 |
| 5.08 -- 1.1 × 1010 |
| 4 4.86 4.99 7.8 × 109 |
| 5.97 6.08 5.6 × 109 |
| 7 5.26 5.39 7.7 × 109 |
| -- -- -- |
| 10 5.62 -- 6.7 × 109 |
| 6.29 6.28 2.0 × 106 |
| ______________________________________ |
| a SCC = simulated Cheddar cheese activity test described in the New |
| Zealand Journal of Dairy Technology 4:246, 1969 which is a measurement of |
| the pH attained by culture acid production during simulated Cheddar chees |
| production. |
| b cfu/ml = viable colony forming units or cells per ml. |
The cells were more viable due to the controlled release of the method of the present invention as can be seen from Table III.
A growth medium was prepared in the same manner as set forth in Example 4. Ten grams (10 g) of pan-coated sodium carbonate granules coated with ethyl cellulose (20% Ethocel® 45) were added to the medium which was then inoculated with 1% by volume Streptococcus lactis 131 B1 (Oregon State University) containing 108 cells per ml and the same fermentation conditions were applied as described in Example 4. A second fermentation was performed in the same manner except the controlled release granules were omitted. A third fermentation was done exactly like the other two except uncoated sodium carbonate was added in place of the granules.
As in Example 4, the cells from completed fermentation of Table IV were stored at 21°C for 10 days, during which the activity and cell numbers were determined. The results are tabulated below in Tables IV, V, VI and VIA. The third fermentation (control b) produced no significant results and impaired or killed large numbers of bacteria.
| TABLE IV |
| ______________________________________ |
| Observed pH |
| Fermentation Time |
| Controlled Release |
| (Hours) Alkali Controla |
| Controlb |
| ______________________________________ |
| 0 6.4 6.5 10.1 |
| 2 6.7 6.5 -- |
| 4 7.0 6.4 -- |
| 6 6.6 5.9 -- |
| 8 5.7 4.9 -- |
| 10 5.0 4.7 -- |
| 12 5.0 4.7 -- |
| 16 5.0 4.7 10.1 |
| ______________________________________ |
| a without alkali |
| b alkali without controlled release |
| TABLE V |
| ______________________________________ |
| Activity and Cell Population Data |
| Controlled Release |
| Activity Controla |
| Controlb |
| ______________________________________ |
| 6 hr at 30°C |
| 4.92 5.11 6.55 |
| activity (pH) |
| cfu/ml 9.2 × 109 |
| 3.3 × 109 |
| 3.1 × 106 |
| ______________________________________ |
| TABLE VI |
| ______________________________________ |
| Controlled Release |
| Storage Time Activity Cell Count |
| (days) (6 hr - 30°C pH) |
| cfu/ml |
| ______________________________________ |
| 0 4.92 9.2 × 109 |
| 2 -- -- |
| 4 5.17 7.0 × 109 |
| 7 5.23 5.6 × 109 |
| 10 5.39 5.4 × 109 |
| ______________________________________ |
| TABLE VI-A* |
| ______________________________________ |
| Storage Time Control Activity |
| Cell Count |
| (days) (6 hr - 30°C pH) |
| cfu/ml |
| ______________________________________ |
| 0 5.11 3.3 × 109 |
| 2 5.73 2.2 × 109 |
| 4 6.12 3.6 × 108 |
| 7 6.27 1.4 × 108 |
| 10 6.30 1.1 × 107 |
| ______________________________________ |
| *Control "a" cultures |
Sodium carbonate tablets (1 gram each) were pancoated with ethyl cellulose (Ethocel® 45) dissolved in methylene chloride/acetone (50/50 by volume) with a coating by weight varying around 1% (0.85-1.25%). The tablets (10 per 700 ml) were then added to the Actilac® growth medium of Example 1 which was inoculated with Streptococcus cremoris 134 (Oregon State University) at 108 cells per ml. The pH was monitored in comparison to control medium without added tablets. The pH data after 16 hours at 27°C were as shown in Table VII.
| TABLE VII |
| ______________________________________ |
| % Coating |
| pH |
| ______________________________________ |
| * 4.9 |
| .85 5.5 |
| .95 5.5 |
| 1.05 5.1 |
| 1.15 5.4 |
| 1.25 5.2 |
| ______________________________________ |
| *no added tablets Actilac ® control |
Tablets coated with 0.85% ethyl cellulose were used in a second fermentation in order to determine the number and activity of cells generated. After 16 hours at 27°C, the control Actilac® culture had a pH of 4.5 while the tablet-coating culture had a pH of 5.0 The control and controlled release cultures were stored at about 25°C (ambient temperatures) and tested for acid-producing activity (pH achieved from 1% by volume inoculation into 11% by weight nonfat milk incubated 6 hours at 30°C) and cell numbers. During the 10 day storage period, the pH of the controlled release culture was adjusted to pH 5.0 with sterile 5 N sodium carbonate at daily intervals. The results were as shown in Table VIII.
| TABLE VIII |
| ______________________________________ |
| Control Controlled Release |
| Days pH cfu/ml pH cfu/ml |
| ______________________________________ |
| 0 5.0 2.2 × 108 |
| 4.8 2.7 × 108 |
| 2 5.9 1.4 × 108 |
| 5.4 2.2 × 108 |
| 4 6.4 1.3 × 106 |
| 5.5 2.2 × 108 |
| 8 --* -- 6.1 2.5 × 108 |
| 10 --* -- 6.2 3.1 × 108 |
| ______________________________________ |
| *cells inactive |
These data show that the cell population produced with the neutralizing agent tablets was stable for 10 days and that activity was extended significantly beyond the control since they were not impaired by the lower pH.
A growth medium was prepared by mixing 21 g of whey powder with 3.5 g of yeast extract, 1.0 g of citric acid and 20 g of magnesium phosphate tribasic which is an essentially water insoluble neutralizing agent. This mixture was suspended in 700 ml of water, heated to 85°C for 45 minutes, cooled to 27°C and inoculated with a lactic acid-producing microorganism which was Streptococcus lactis 134 (Oregon State University) at 108 cells per ml. A control medium without the citric acid and the magnesium phosphate tribasic was treated in a similar manner. Another medium containing 21 g of whey powder with 3.5 g of yeast, 2 g of citric acid and 10 g of calcium carbonate which is also an essentially water insoluble neutralizing agent was treated similarly. The pH values of the growth media after inoculation are shown in Table IX. The citric acid was used to initially adjust the pH.
| TABLE IX |
| ______________________________________ |
| pH Treatment 1 pH Treatment 2 |
| Time (magnesium phosphate |
| Calcium |
| (Hours) |
| tribasic) carbonate pH-control |
| ______________________________________ |
| 0 7.00 6.52 6.03 |
| 2.25 7.01 6.63 6.08 |
| 3.33 7.07 6.67 6.16 |
| 4.33 7.03 6.69 6.06 |
| 8.33 6.78 6.22 5.72 |
| 9.00 6.65 6.15 5.58 |
| 9.50 6.59 6.06 5.48 |
| 10.00 6.50 5.97 5.41 |
| ______________________________________ |
The starter culture for the products containing calcium carbonate or magnesium phosphate had increased activity relative to the control. Similar fermentations using 2, 3 or 4 times as much calcium carbonate or magnesium phosphate gave very similar results.
Other fermentations repeating Examples 1 to 7 using anion/cation exchange resins as well as different amounts of sodium phosphate monobasic, ammonium phosphate monobasic, ammonium citrate dibasic or disodium citrate as the insoluble or insolubilized neutralizing agents were performed and the acid produced was at least partially neutralized and the activity of the resultant bacteria was higher than from the control fermentations.
Actilac® growth medium was placed in 700 ml of water to achieve a solids level of 11% by weight, heated to 85°-90°C for 45 minutes, cooled to 27°C and inoculated with a one percent (1%) by volume with a lactic streptococcal (Streptococcus cremoris 134 ((Oregon State University) of 108 cells per ml) starter culture. Fermentation was continued for 12 hours at 27°C, when a pH of 4.6 was achieved, and then sterile 5 N sodium carbonate was added to raise the pH to 7∅ The fermentation was allowed to continue for an additional 4 hours (pH was 5.4) for a total fermentation time of 16 hours. Samples were taken at 8 hours and at 16 hours, stored at 25°C and at 5°C and analyzed daily for stability as determined by acid-producing activity and number of viable bacteria present per milliliter. These data, shown in Tables X and XI below, allowed activity comparison of controlled-release base produced cells with cells produced when alkali was added late in the fermentation.
| TABLE X |
| ______________________________________ |
| Data on cells sampled at 8 hours |
| Stor- |
| age Activity (pH) |
| Time 6 hr - 30°C |
| SCC cfu/ml |
| (Days) |
| 5°C |
| 25°C |
| 5°C |
| 25°C |
| 5°C |
| 25°C |
| ______________________________________ |
| 0 4.88 4.88 5.10 5.10 2.1 × 108 |
| 2.1 × 108 |
| 1 5.65 5.62 5.99 6.03 5.4 × 107 |
| 1.5 × 108 |
| 2 5.83 6.24 6.09 6.31 3.5 × 107 |
| 2.2 × 107 |
| 4 5.96 6.37 -- -- 3.9 × 107 |
| 1.8 × 106 |
| 7 6.09 6.37 -- -- 4.9 × 107 |
| <4.0 × 104 |
| ______________________________________ |
| TABLE XI |
| ______________________________________ |
| Data on cells sampled at 16 hours |
| Storage |
| Activity (pH) |
| Time 6 hr - 30°C |
| SCC cfu/ml |
| (Days) 5°C |
| 25°C |
| 5°C |
| 25°C |
| 5°C |
| 25°C |
| ______________________________________ |
| 0 4.74 4.74 4.90 4.90 4.9 × 108 |
| 5.6 × 108 |
| 1 5.03 4.90 5.49 5.22 2.8 × 108 |
| 6.4 × 108 |
| 2 5.95 5.12 6.18 5.46 9.1 × 107 |
| 5.0 × 108 |
| 4 6.26 6.22 -- -- 1.9 × 107 |
| 1.3 × 108 |
| 7 6.30 6.38 -- -- 1.3 × 107 |
| 1.6 × 106 |
| ______________________________________ |
Comparison of these data to those generated for Example 4 illustrate the advantages of the timed-release pH control methods over that of adding alkali late in the fermentation to neutralize acid present. The cells are acid damaged.
Starter culture was prepared in a medium containing 3.5% whey powder, 0.5% yeast extract, 3.6% magnesium phosphate tribasic, 0.5% dibasic ammonium citrate and 0.5% tribasic sodium citrate dihydrate as in the first treatment of Example 7 and a control culture was prepared by inoculation of 11% solids nonfat milk followed by incubation at 27°C also for 16 hrs to a pH of 4.4 which damaged the cells due to acidity. The two cultures were compared for acid-producing activity, viable cell counts and for acid production during the manufacture of Cheddar cheese using acceptable industry procedures but with only a 0.5% by volume (5×107 cells per ml) inoculum as compared to the 1 to 3% by volume customarily used. For the culture produced under the influence of the water insoluble or insolubilized neutralizing agent, the pH value by the 6 hour 30°C activity test was 4.75 and for the SCC test it was 4.89 and the viable cell count was 1.1×109 cfu per milliliter. For the control culture these pH values were 4.94 and 5.30, respectively and the viable cell count was 1.6×109. The culture produced with pH-dependent released neutralizing agent was noticeably more active during cheese making as shown by the data in Table XII.
| TABLE XII |
| ______________________________________ |
| Titratable Acidity |
| Step Test Culture |
| Nonfat Milk Culture |
| ______________________________________ |
| Raw Milk 0.15 0.15 |
| After 30 minute |
| 0.15 0.15 |
| ripening |
| After cutting |
| 0.10 0.10 |
| After washing |
| 0.12 0.10 |
| Start cheddaring |
| 0.24 0.18 |
| Half cheddared |
| 0.37 0.20 |
| End of cheddaring |
| 0.47 0.30 |
| ______________________________________ |
| *pH controlled culture prepared as in the first treatment of Example 7. |
| These data show the superior acidproducing activity of the bacterial |
| starter cultures produced by the present invention. |
Another medium formulation containing magnesium phosphate tribasic was evaluated for growth, pH maintenance and phage inhibition. The medium contained by weight 1.5% magnesium phosphate tribasic, 1.5% ammonium phosphate dibasic, 1.5% tribasic sodium citrate dihydrate, 3.5% sweet whey powder and 0.5% yeast extract. It was heat treated as in Example 7. Evaluation was made in comparison to nonfat dry milk (11% solids), a leading phage inhibitory medium (HL-100; Chr. Hansen's Laboratory, Inc., Milwaukee, Wisconsin) and the continuous whey neutralization process now practiced in industry and described by Richardson (Dairy and Ice Cream Field 161(9): 80A-80D (1978)). Fermentation was at 21°C The continuous neutralization process uses 27°C in industry, however this is not a significant difference. One percent inoculum was made with Streptococcus cremoris 205 (Oregon State University) and the homologous phage T189 (105 phages per ml) was used to evaluate the phage inhibition. Table XIII below shows the superior growth promoting, phage inhibitory and active cell generating properties of the medium containing magnesium phosphate as the water insoluble neutralizing agent. Also shown in Table XIII are data indicating that the cells produced by the controlled release of the neutralizing agent are not in an injured state and therefore maintain acid producing activity for at least 24 hours at ambient room temperature.
| TABLE XIII |
| __________________________________________________________________________ |
| Acid Producing Activitya |
| Cell Numbers |
| 0 hr 24 hr b |
| (cfu/ml) Phage Inhibition |
| Δ4 |
| Δ6 |
| Δ4 |
| Δ6 |
| 0 hr 24 hr |
| Phage added/ml |
| Phage recovered |
| __________________________________________________________________________ |
| NDW(11% W/W) |
| 0.44 |
| 1.05 |
| 0.34 |
| 0.69 |
| 2.0 × 109 |
| 2.0 × 109 |
| 9.2 × 104 |
| 4.2 × 109 |
| HL 100(11.5% W/W) |
| 0.69 |
| 1.42 |
| 0.46 |
| 1.05 |
| 2.9 × 109 |
| 2.8 × 109 |
| 8.7 × 104 |
| 2.5 × 105 |
| Continuous 0.99 |
| 1.60 |
| 0.72 |
| 1.41 |
| 7.2 × 109 |
| 9.2 × 109 |
| 9.2 × 104 |
| 6.4 × 107 |
| Neutralization |
| 1.5/1.5/1.5 Medium |
| 1.14 |
| 1.69 |
| 0.75 |
| 1.44 |
| 1.0 × 1010 |
| 8.5 × 109 |
| 8.7 × 104 |
| 2.3 × 103 |
| __________________________________________________________________________ |
| a 6 hr30°C activity test with pH readings at 4 hr and 6 |
| hr(Δ4 and Δ6 are changes in pH after 4 and t hrs. |
| respectively). |
| b activity after storage for 24 hours at room temperature |
The magnesium phosphate-containing medium was also evaluated for inhibition of other lactic streptococcal bacteriophage. Data showing that the controlled release medium prevents phage replication and causes a reduction in phage titer appear in Table XIV.
| TABLE XIV |
| ______________________________________ |
| Phage/Host φ added/ml |
| φ recovered/ml |
| ______________________________________ |
| T189/205 8.7 × 104 |
| 2.3 × 103 |
| Ml8 /ML8 * |
| 3.1 × 104 |
| 4.3 × 103 |
| hp/HP 4.4 × 104 |
| <1.0 × 103 |
| h2 /H2 3.7 × 104 |
| <1.0 × 103 |
| C2/C2* 5.9 × 104 |
| <1.0 × 103 |
| ______________________________________ |
| * Streptococcus lactis strains; other strains are Steptococcus |
| cremoris "φ" means phage and all are available at Oregon State |
| University. |
The specific improvements of the present invention thus include: (1) the use of the essentially water insoluble or temporarily insolubilized acid neutralizing agents which may be incorporated initially or added at any time to fermentation systems such that the agent is available at a rate to at least partially neutralize acid produced during the growth of fermentation process. (2) The use of a mixture of bulk starters including essential growth chemicals with powdered, pelleted, tableted or granulated essentially insoluble or insolubilized neutralizing agents, with sufficient amounts of acidic materials to obtain the desired initial pH for use as culture media for bacterial starter production for use in the preparation of cheese and for other food or even nonfood products prepared by fermentation. (3) The use of the essentially water insoluble or temporarily water insolubilized neutralizing agents in the dry form or after dissolution in bacterial cultures.
Sandine, William E., Ayres, James W.
| Patent | Priority | Assignee | Title |
| 5364641, | Jun 08 1989 | Roquette Freres | Process for manufacturing dairy products |
| Patent | Priority | Assignee | Title |
| 1190827, | |||
| 1913346, | |||
| 2549765, | |||
| 2845354, | |||
| 3098016, | |||
| 3133862, | |||
| 3242051, | |||
| 3622350, | |||
| 3639306, | |||
| 3715276, | |||
| 3792171, | |||
| 3937798, | Aug 17 1970 | Fuji Photo Film Co., Ltd. | Method of controlling hydrogen ion concentration by microcapsules |
| 3943063, | Dec 30 1971 | Toyo Jozo Company, Ltd. | Preparation of microcapsules |
| 3949094, | Jul 31 1974 | DURKEE INDUSTRIAL FOODS CORP | Condiment-treating process and product |
| 3949096, | Jul 31 1974 | DURKEE INDUSTRIAL FOODS CORP | Spray drying process for encapsulation of condiment particles |
| 3952110, | Jul 03 1973 | General Foods, Limited | Dry mix for coating foods |
| 3959499, | Dec 23 1974 | The United States of America as represented by the Secretary of the Army | Leavening composition and storage stable cake mixes made therewith |
| 3976794, | Jun 03 1974 | DURKEE INDUSTRIAL FOODS CORP | Encapsulation of sugar and its use in sweetened coconut |
| 3983913, | Jun 03 1975 | Emco Wheaton Inc. | Vapor recovery and vent signal system |
| 3996156, | May 28 1973 | Fuji Photo Film Co., Ltd. | Production of microcapsules |
| 4004039, | Dec 03 1973 | General Foods Corporation | Sweetening composition and process therefor |
| 4021304, | Apr 19 1973 | Dainippon Sugar Manufacturing Co., Ltd.; Dainippon Ink and Chemicals, Incorporated | Process for continuous cultivation of protein-producing microorganisms |
| 4053642, | May 02 1972 | STICHTING NEDERLANDS INSTITUUT VOOR ZUIVELONDERZOEK, KERNHEMSEWEG 2, P O BOX 20, 6710 BA EDE GLD , THE NETHERLANDS | Starter culture production |
| 4115199, | May 04 1977 | CHR HANSEN, INC | Preparation of culture concentrates for direct vat set cheese production |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Aug 01 1983 | State of Oregon, by and through the Oregon State Board of Higher | (assignment on the face of the patent) | / |
| Date | Maintenance Fee Events |
| Date | Maintenance Schedule |
| Feb 04 1989 | 4 years fee payment window open |
| Aug 04 1989 | 6 months grace period start (w surcharge) |
| Feb 04 1990 | patent expiry (for year 4) |
| Feb 04 1992 | 2 years to revive unintentionally abandoned end. (for year 4) |
| Feb 04 1993 | 8 years fee payment window open |
| Aug 04 1993 | 6 months grace period start (w surcharge) |
| Feb 04 1994 | patent expiry (for year 8) |
| Feb 04 1996 | 2 years to revive unintentionally abandoned end. (for year 8) |
| Feb 04 1997 | 12 years fee payment window open |
| Aug 04 1997 | 6 months grace period start (w surcharge) |
| Feb 04 1998 | patent expiry (for year 12) |
| Feb 04 2000 | 2 years to revive unintentionally abandoned end. (for year 12) |