FREEZE-DRIED PROBIOTIC FOODSTUFFS

Abstract

Compositions wherein foodstuffs act as carriers of freeze-dried probiotics provide new means for administration of probiotics. The products that are easily shipped may be eaten as purchased or mixed with other ingredients to provide variety in the diet while administering beneficial probiotic organisms to the gastrointestinal tract.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. A method of manufacturing a composition, wherein the method comprises: 1) applying or mixing a probiotics composition onto or into a food, then 2) freeze-drying the product of step 1.

9. The method of claim 8, wherein, in step 1, the probiotics composition is a probiotic-containing powder or a probiotic-containing solution.

10. The method of claim 8, wherein the food is a dried vegetable, meat, fruit or nuts and the probiotics composition contains freeze-dried probiotic organisms, wherein the composition is a powder or a solution which is applied to said food.

11. The method of claim 8, wherein the food is a dairy product and the probiotics composition is mixed into said dairy product.

12. The method of claim 8, wherein the product of step 2 is then freeze-dried.

13. The method of claim 10, wherein the food, before step 1, has been subjected to at least one process chosen from salt curing, heating and pickling prior to said applying the probiotics composition.

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic representation of a transverse cross section of preparation of a probiotic freeze-dried food.

[0017] FIG. 2 is a flow diagram of several steps that may be used in practice of the invention.

[0018] Meat pre-treatment: Beef meat or chicken livers were rinsed with large amounts of water, dried with paper towels, cut into inch to 2 inch cubes, heat seared in olive oil. Seared cubes were removed and placed on paper towels and cooled to room temperature. Cooled cubes were treated with probiotic powder or probiotic solution as detailed below, and mixed homogenously and then placed trays for freeze-drying.

[0019] Alternatively, the meats may be treated with salt, freeze dried or subjected to antioxidant treatment to destroy harmful organisms,

[0020] Coating with probiotic powder (hereinafter 5051 powder): Pieces of foodstuff (e.g. seared and cooled cubes as described above) were placed into containers with probiotic powder (40 g to 80 g P. acidilactici NRRL B-50517 powders, 10 billion CFU/g for 200 g to 1 kg of foodstuff). The pieces were rolled in probiotic-containing powder in order to coat the pieces, then transferred to trays for freeze-drying. In some instances the trays were covered with parchment paper.

[0021] Coating with probiotic solution (hereinafter 5051 solution): Pieces of foodstuff (e.g. seared and cooled cubes as described above) were placed into containers with a Pediococcus acidilactici NRRL B-50517 solution [mixed 20 g of Pediococcus fermentative probiotics of freeze-dried powders (catalogue number PA-5051FD, Imagilin Technology, LLC) with 80 ml water]. The pieces were mixed in the solution for 45 secs to one minute then transferred to trays for freeze-drying. Sometimes the trays were covered with parchment paper. Alternatively, pieces of food may be coated with probiotic fermentative culture or yogurt containing the probiotics.

[0022] Calculation of CFU: in some examples of the invention 0.2 g to 0.4 g probiotic bacteria coated foodstuff was transferred into 5 ml saline solution, and mixed well. A series of dilutions was prepared and plated in 100 ul aliquots onto MRS plates and incubated overnight. Colonies were then counted. The CFU determination was made before and after freeze-drying and/or at other steps in the process.

[0023] Referring with particularity to the drawings, FIG. 1 is a flow chart of the processes of the invention that may be used to prepare the foodstuffs for application of freeze dried probiotics to the foodstuffs. Referring to the drawings, (1) is the entrance of food stuffs into the process of preparation. The cleaning process may be initiated using non-heat treatment (2) or heat treatment (3). Foodstuffs which have been subjected to non-heat treatment (2) may then be frozen 4, or may not be frozen (5). In another stream, foodstuffs upon entering the heat treatment stream (3) may later be non-frozen (6) or frozen (7). At the end, all foodstuffs from the preparatory process are (8) then are passed on to the process for addition of the probiotic-containing materials. The probiotics may be applied as yogurt probiotic compositions (9), as fermented growth probiotic compositions (10), as probiotic-containing solutions (11) or as probiotic powder compositions (12). All products from streams 9, 10, 11 and 12 now pass through the freeze-drying process (13) producing freeze-dried probiotic products (14).

[0024] FIG. 2 is another flow chart summary of the process. The foodstuffs are cleaned (15), then may be heat treated (16) or may undergo treatment that does not involve heating (17)). The products of stream 16 may then be frozen (21), but need not be frozen (20). Similarly, products of stream 17 may pass on in the production process after being frozen (19) or without being frozen (18). The stream of prepared foodstuffs products (22) then may pass on to the process for application or incorporation of the freeze dried probiotic. The application or incorporation of probiotics may be in the form of yogurt prepared products (23), fermented growth probiotics (24). Probiotic solutions (25) or probiotic powders (26). All of the products from streams 23, 24, 25, and 26, regardless of means of application of the probiotics, then undergo freeze drying (27) to provide probiotic freeze-dried final products (28).

Example 1

Detection of Live Probiotics from Pediococcus Freeze-Dried Beef Meat and Chicken Livers

[0025] Heat-seared beef and/or chicken livers were prepared for coating with probiotic bacteria. Beef meat or chicken livers were subjected to meat pre-treatment as described above, then coated with either 5051 powder or 5051 solution (see above) and freeze dried. CFU/g were determined after freeze-drying. Results are summarized in table 1.

TABLE-US-00001 TABLE 1 recovery of viable bacteria from cooked meat products after freeze-drying Substrate NRRL B-50517 CFU/g Beef meat 5051 powders 9.03 10.sup.8 5051 solutions 2.24 10.sup.8 Chicken Livers 5051 powders 1.68 10.sup.9 5051 solutions 1.18 10.sup.8

[0026] Results presented in table 1 indicate that bacteria remained viable after freeze-drying on heat-treated beef and/or chicken livers.

Example 2

Applications of Lactobacillus fermentum and Pediococcus acidilactici NRRL B-50517

[0027] In order to determine whether freeze-drying can be applied to bacteria from strains other than Pediococcus acidilactici NRRL B-50517 a similar preparation method using probiotic Lactobacillus fermentum was used. Both live Lactobacillus fermentum and P. acidilactici were detected from Lactobacillus freeze-dried sliced apples and Pediococcus freeze-dried apples at room temperature or those treated at 65 C. for 10 minutes. Survival of P. acidilactici NRRL B-50517 through freeze-drying process is 540 times better than Lactobacillus fermentum, and 2,850 times better once the probiotic freeze-dried products were treated by elevated temperature at 65 C. for 10 minutes (table 2). These results demonstrated the similar preparation methods are applicable using different probiotic bacteria. However, the survival rates of Lactobacillus fermentum is much less (about 1,000 folds less) than those of P. acidilactici NRRL B-50517

[0028] Results are summarized in table 2.

TABLE-US-00002 TABLE 2 Detection of live probiotics either from Lactobacillus probiotic freeze- dried apples* or from Pediococcus probiotic freeze-dried apples** Numbers of Numbers of Lactobacillus Pediococcus fermentum acidilactici NRRL Treatment of (CFU/g) from B-50517 (CFU/g) from probiotic freeze- probiotic freeze- probiotic freeze- dried apples dried Gala apples.sup.1 dried Gala apples.sup.1 Room temperature 4.25 10.sup.5 2.30 10.sup.8 65 C. 10 minutes 1.00 10.sup.5 2.85 10.sup.8 *Apples were cut into inch slices, then those pieces were cut into halves. Apple slices were transferred into the Lactobacillus fermentum solution [mixed 20 g of Lactobacillus fermentative probiotics for freeze-dried powders 10 billion CFU/g, with 80 ml water], then mixed with the probiotic solution for 45 secs to a minute and then transferred Lactobacillus coated apple on the freeze-drying trays covered with parchment paper and freeze dried. **Cut apples into inch slices, then cut those into halves. Transferred apple slices into the Pediococcus acidilactici NRRL B-50517 solution [mixed 20 g of Pediococcus fermentative probiotics for freeze-dried powders 10 billion CFU/g with 80 ml water], mixed the sliced apples and probiotic solution for 45 secs to a min, transferred Pediococcus coated apple on the freeze-drying trays covered with parchment paper and freeze dried. .sup.1Measured 0.2 g to 0.4 g of Lactobacillus probiotic freeze-dried sliced apples or Pediococcus probiotic freeze-dried sliced apples, transferred the sliced apple into 5 ml saline solution, and mixed well. Performed series of dilutions, and plated 100 ul of desirable diluted solution onto MRS plates and incubated overnight, then counted the colonies.

Example 3

Increasing Loading of Bacteria in Coating

[0029] In order to examine the possibility of increasing the amount of CFUs per gram in freeze-dried apples, we increased the loading amount prior to freeze-drying.

[0030] This is important because when one takes higher amounts of live probiotics, the beneficial effects are observed more quickly, especially for those suffering from digestive disorders. Appling of higher amounts of P. acidilactici NRRL B-50517 onto the products prepared by the freeze-dried process resulted in proportionally higher amounts of live Pediococcus detected. These results demonstrate the possibility of making probiotic freeze-dried products with any desired amount of live bacteria depending on the concentrations applied before freeze-drying. Results are summarized in table 3.

TABLE-US-00003 TABLE 3 Detection of live probiotics from Pediococcus freeze-dried apples using different amounts of P. acidilactici NRRL B-50517 Amounts of P. acidilactici NRRL B-50517 Numbers of P. acidilactici NRRL B-50517 (CFU) per kg of (CFU/g)** from probiotic freeze-dried Gala Fuji apples (fresh weight)* apples 4 10.sup.12 4.85 10.sup.9 4 10.sup.11 6.85 10.sup.8 4 10.sup.10 6.38 10.sup.7 *Cut apples into inch thick slices, then cut those into halves, then transferred apple slices into the Pediococcus acidilactici NRRL B-50517 solution [mixed 20 g of different concentration of Pediococcus fermentative probiotics for freeze-dried powders with 80 ml water], mixed the sliced apples and probiotic solution for 45 secs to a min, transferred Pediococcus coated apple on the freeze-drying trays covered with parchment paper and freeze dried. **Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated overnight and then counted the colonies.

Example 4

Pretreatment of Apples has No Apparent Effect on Probiotic Viability of Probiotic Freeze-Dried Food.

[0031] In order to determine the effects of boiling and/or freezing foodstuffs on the ability to make probiotic coated freeze-dried foodstuffs an experiment was conducted in apples. Results summarized in table 4 indicate that P. acidilactici NRRL B-50517 is able to survive freeze-drying when applied as a coating to frozen apples, heat treated apples, and heat treated and frozen apples. More important, similar viabilities of probiotics were obtained from frozen apples, heat-treated apples, and heat-treated and frozen apples. This indicates that P. acidilactici NRRL B-50517 can survive the freeze-drying process from on foodstuffs subjected to various pre-treatments.

TABLE-US-00004 TABLE 4 Detection of live probiotics from Pediococcus freeze-dried apples using different pretreatment of apples followed by treating with probiotic solutions Numbers of P. acidilactici Pretreatment of apples* NRRL B-50517 (CFU/g) ** Boiled apples.sup.1 2.60 10.sup.7 Frozen apples.sup.2 3.40 10.sup.7 Boiled, frozen apples.sup.3 8.93 10.sup.7 *.sup.1Boiled apples-transferred the sliced apples to the 90 C. heated saline solutions for 30 seconds to 1 minute, took apple slices out and cooled to room temperature, then coated with probiotic solution 5051 as described hereinabove. .sup.2Frozen apples-transferred the sliced apples to 10% sucrose solution, mixed uniformly, transferred to 20 C. freezer to freeze the apples overnight to a week, took frozen apple slices then coated with probiotic solution 5051 as described hereinabove. .sup.3Boiled then frozen apples-transferred the sliced apples to the 90 C. heated saline solutions for 30 seconds to 1 minute, took apple slices out and cooled to room temperature, transferred the sliced apples to 10% sucrose solution, mixed uniformly, transferred to 20 C. freezer to freeze the apples overnight to a week, took boiled, frozen apple slices then coated with probiotic solution 5051 as described hereinabove. *Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the sliced apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies.

Example 5

Powder Vs. Solution as Coating Agent

[0032] In order to determine whether there is a significant difference between coating with powder or solution, we conducted a direct comparison using multiple foodstuffs as substrates. One of the motivations for this experiment is that some industrial manufacturing process may prefer spraying or coating the powders onto the products directly. In this experiment either Pediococcus solution or Probiotic powder was applied directly onto blueberry, sliced strawberry, sliced green bean and sliced sweet potato followed by the freeze-drying to see the survival of P. acidilactici NRRL B-50517. CFU were then determined from the freeze-dried products.

[0033] Results are summarized in table 5. Results indicate that high amounts of viable Pediococcus bacteria were present in freeze-dried products prepared using Pediococcus probiotic powders directly.

TABLE-US-00005 TABLE 5 Detection of live probiotics from Pediococcus freeze-dried fruits or vegetables using probiotic powders or probiotic solution * Numbers of P. acidilactici NRRL B- Type of 50517 (CFU/g) ** P. acidilactici from probiotic freeze- Water substrate NRRL B-50517** dried products Activity Blueberry Powders1 4.90 10.sup.8 0.32 Solution.sup.2 6.25 10.sup.7 0.34 Strawberry Powders.sup.1 7.90 10.sup.8 0.25 Solution.sup.2 1.00 10.sup.8 0.18 Green Bean Powders.sup.1 6.00 10.sup.8 0.31 Solution.sup.2 1.25 10.sup.8 0.20 Sweet potato Powders.sup.1 4.00 10.sup.8 0.28 Solution.sup.2 1.25 10.sup.8 0.26 .sup.1Placed blueberry, sliced strawberry, sliced green bean, sliced and cooked sweet potato into containers with 5051 powder as described above. Pediococcus coated blueberry, strawberry, green bean and sweet potato were placed on separate trays covered with parchment paper and freeze-dried. .sup.2Transferred pieces of blueberry, sliced strawberry, sliced green bean, sliced and cooked sweet potato into 5051 solution as described hereinabove. Pediococcus coated blueberry, sliced strawberry, sliced green bean, sliced and cooked sweet potato were placed on separate trays covered with parchment paper and freeze-dried.

Example 6

Use of Probiotic Fermentation Culture to Coat Foodstuffs

[0034] An additional experiment was conducted in order to determine whether fermentation cultures stored at room temperature for varying amounts of time could serve as a source of probiotic coatings instead of 5051 solution or 5051 powder. Results are summarized in table 6.

[0035] Preparing probiotic bacteria directly from cultures and applying to foodstuffs before freeze-drying can increase efficiency and/or reduce cost of preparing probiotic coated freeze-dried products.

[0036] In order to examine this possibility, P. acidilactici NRRL B-50517 fresh cultures were prepared in fermenters, harvested probiotics, and resuspended in a cryoprotectant solution. These probiotic solutions were applied to sliced Gala apple and sliced Fuji apples then freeze-dried. We successfully detect the live probiotic from these probiotic Fuji apples and gala apples.

[0037] Results summarized in table 6 show the viability of P. acidilactici NRRL B-50517 was not appreciably decreased even after 64 days stored at room temperature. This demonstrates that fresh prepared Pediococcus probiotics can be applied to the materials for preparation of probiotic freeze-dried products, and these probiotic freeze-dried products are stable at room temperature storage.

TABLE-US-00006 TABLE 6 Detection of live probiotics from Pediococcus freeze-dried apples using Pediococcus prepared fresh by fermented cultures after stored at room temperature* Numbers of P. acidilactici Numbers of P. acidilactici Days NRRL B-50517 (CFU/g)** NRRL B-50517 (CFU/g)** stored at room from probiotic freeze-dried from probiotic freeze-dried temperature Gala apples Fuji apples 1 2.00 10.sup.8 2.50 10.sup.8 8 2.90 10.sup.8 3.35 10.sup.8 24 1.30 10.sup.8 1.10 10.sup.8 40 1.78 10.sup.8 1.33 10.sup.8 50 2.00 10.sup.8 5.50 10.sup.8 64 1.68 10.sup.8 1.70 10.sup.8 *Cut apples into inch slices, then cut those into halves. Transferred apple slices into the fresh prepared Pediococcus acidilactici NRRL B-50517 harvested from fermenters. Preparation of P. acidilactici NRRL B-50517 cultures harvested from fermenter-transferred 10 well separated to 10 ml MRS broth in 50 ml falcon tube and incubated at 45 C. overnight, transferred the 10 ml overnight bacterial cultures to 2 liters of fermenters and incubated at 100 rpm, 45 C. overnight, harvested the overnight bacterial cultures by centrifugation, added 150 ml solution of 7% sucrose, 7.5% lecithin to resuspend the bacterial cultures homogenously. After mixed the sliced apples with Pediococcus fermented cultures solution, placed the apple slices on the tray evenly, proceed the freeze-dried process until it completed. **Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples stored at room temperature at desirable time, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies.

Example 7

Coating with Yogurt

[0038] The most common application of probiotics is preparation of yogurt from milks. P. acidilactici NRRL B-50517 monoculture can be used to make yogurt. Unlike conventional yogurt starter culturesStreptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus that die after yogurt formation, P. acidilactici NRRL B-50517 maintains viability after yogurt formation months in the refrigerator. This suggested yogurt can be a source of Pediococcus probiotics to apply to the foodstuffs before freeze-drying. In order to test this hypothesis, yogurt was prepared using P. acidilactici NRRL B-50517 and applied directly onto sliced apples followed by freeze-drying to prepare Pediococcus probiotic freeze-dried apples.

[0039] Results presented in table 7 show live P. acidilactici NRRL B-50517 from Pediococcus yogurt treated freeze-dried apples. These results suggest an alternative method to introduce Pediococcus probiotics onto freeze-dried foodstuffs and/or provide a way to introduce dairy products onto other foodstuffs.

TABLE-US-00007 TABLE 7 Detection of live probiotics from Pediococcus freeze-dried apples mixed with yogurt prepared by P. acidilactici NRRL B-50517* Freeze-dried apples mixed with yogurt Prepared from Numbers of P. acidilactici NRRL B-50517 P. acidilactici (CFU/g) from freeze-dried Gala apples mixed NRRL B-50517 with Pediococcus made yogurt** Sample 1 2.58 10.sup.7 Sample 2 4.15 10.sup.7 *Cut apples into slices, then cut those into halves. Transferred apple slices into the yogurt prepared by P. acidilactici NRRL B-50517, mixed the apple slice with Pediococcus yogurt homogenously, transferred Pediococcus yogurt coated apple on the freeze-drying trays covered with parchment paper and freeze-dried. Preparation Pediococcus yogurt: **Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies.

Example 8

Freeze-Dried Substrate, Coated and Freeze-Dried Again

[0040] In order to examine the possibility of coating a freeze-dried substrate with solution 5051 the freeze-drying again, an experiment was conducted using apples. Results summarized in table 8 indicate a 100-fold decrease in viable bacteria as a result of freeze-drying.

TABLE-US-00008 TABLE 8 Detection of live probiotics from Pediococcus freeze-dried apples by applying probiotics solution onto freeze-dried apples with P. acidilactici NRRL B-50517* and through the 2.sup.nd freeze-dried process. Numbers of P. acidilactici NRRL B-50517 (CFU/g) from multiple freeze-dried Gala apples mixed with Amounts of P. acidilactici NRRL B-50517 P. acidilactici NRRL B- solution spraying onto freeze-dried apples 50517 1 109 CFU probiotic onto per g 2.30 10.sup.7 freeze-dried apple 2 109 CFU probiotic onto per g 4.50 10.sup.7 freeze-dried apple *Cut apples into inch slices, then cut those into halves. Transferred apple slices on the freeze-drying trays covered with parchment paper, freeze-dried, removed the tray, and sprayed Pediococcus probiotic solution 5051 onto freeze-dried apples and freeze-dried again. **Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight, then counted the colonies.

Example 9

Heat Treatment of Probiotic Freeze-Dried Meat Products

[0041] P. acidilactici NRRL B-50517 is shown to be able to survive after high temperature (up to 85 C.), high osmotic stress (example 11 hereinbelow), and low pH treatment (example 10 hereinbelow). It is also more tolerant to the antibiotics treatment (Example 12 hereinbelow).

[0042] A series of experiments was conducted In order to determine whether P. acidilactici NRRL B-50517 maintains desired qualities after freeze-drying.

[0043] After incubating both Pediococcus freeze-dried beef meat and Pediococcus probiotic freeze-dried chicken livers at 65 C. incubator for 30 min, live P. acidilactici NRRL B-50517 is detected from both Pediococcus probiotic beef meat and Pediococcus probiotic chicken livers. Further after treatment these Pediococcus probiotics at 85 C. for 10 min and 30 min after incubated at 65 C. for 30 minutes, we were able to detect 20% to 30% survival of P. acidilactici NRRL B-50517 compared to those stored at room temperature only (Table 9). Results summarized in table 9 indicate that P. acidilactici NRRL B-50517 not only survive through the freeze-dried process but also maintains the unique characteristics of high temperature resistance

TABLE-US-00009 TABLE 9 Survival of Probiotics after elevated temperature treatment of Pediococcus probiotic freeze-dried beef meat and chicken livers Number of P. acidilactici NNRL B- % Treatment* 50517 (CFU/g) ** survival Beef meat Room temperature 2.24 10.sup.8 100% 65 C., 30 min 8.75 10.sup.7 39.11% 65 C., 30 min followed 4.75 10.sup.7 21.23% by 85 C., 10 min 65 C., 30 min followed 5.13 10.sup.7 22.91% by 85 C., 30 min Chicken livers Room temperature 1.18 10.sup.8 100% 65 C., 30 min 4.00 10.sup.7 34.04% 65 C., 30 min followed 4.25 10.sup.7 36.17% by 85 C., 10 min 65 C., 30 min followed 7.50 10.sup.7 63.83% by 85 C., 30 min *Incubated Pediococcus probiotic freeze-dried beef meat or Pediococcus probiotic freeze-dried chicken livers at elevated temperature 65 C. for 30 minutes, 65 C. for 30-minute following by 85 C. for another 10 minutes or by 85 C. for another 30 minutes, then measured the detection of probiotics as described at Table 7. Control was to take the Pediococcus freeze-dried beef meat or the Pediococcus freeze-dried chicken livers at room temperature without being treated at elevated temperature and performed the probiotic assays directly.

Example 10

Low pH Treatment

[0044] In order to demonstrate that probiotic freeze-dried products can survive at the stomach acid, 5051 probiotic freeze-dried apples were placed into water adjusted to pH 7.0, 5.0, 3.0 and 2.0, and incubated at room temperature from 30 minutes to 3 hours. Results summarized in table 10 show there are no detectable viable bacteria when 5051 freeze-dried apples were incubated at pH 3.0 up to 1 and hours. However, there is about 1.0% survival of probiotics when incubated at the pH 2.0 for 30 minutes but no further decrease was detectable up to 3-hour incubation at pH 2.0, room temperature (example 10). These results showed Pediococcus acidilactici NRRL B-50517 can integrated into the food through freeze-dried process, and still maintain the resistance to the acidic environment of the stomach.

TABLE-US-00010 TABLE 10 Detection of live probiotics from Pediococcus freeze-dried apples treated after acidic treatment* 30 min 3 hrs. Sterilized Number of 1 hrs Number of water P. acidilactici Number of P. acidilactici P. acidilactici with NNRL B- NNRL B- NNRL B- different 50517 % of 50517 % of 50517 % of pH (CFU/g)** survival (CFU/g)** survival (CFU/g)** survival pH 7.0 5.50 10.sup.7 100.00% 4.35 10.sup.7 100.00% 6.0 10.sup.7 100% pH 5.0 3.38 10.sup.7 61.36% 3.63 10.sup.7 85.29% 5.25 10.sup.7 87.50% pH 3.0 6.25 10.sup.7 113.64% 6.75 10.sup.7 158.82% 2.38 10.sup.7 39.58% pH 2.0 6.25 10.sup.5 1.14% 2.50 10.sup.5 0.59% 1.49 10.sup.5 1.46% *Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml sterilized water adjusted with HCl to the desirable pH in a 15 ml falcon tube, mixed well, incubated at room temperature for thirty minutes, one and half hours and three hours. After incubation, transferred 100 l solution from the falcon tube, performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies. Numbers were the average of two repeated experiments

Example 11

Osmotic Stress

[0045] The mammalian digestive system contains bile salts and stomach acids that can interfere with the ability of probiotic bacteria to reach relevant portions of the GI tract to exert beneficial effects. In order to learn if 5051 freeze-dried products can resist high salt treatment, 5051 freeze-dried apples were incubated in water with concentrations of sodium chloride varying from 0.1% (saline), 1.0%, 5.0%, 10.0% and 20.0% for 30 minutes, 1 hour, 2 hours and overnight at room temperature. Results summarized in table 11 show no detectable viability difference in P. acidilactici NRRL B-50517 from 0.1% to 20% NaCl solution up to 2-hour incubation at room temperature. Even after overnight incubation at the 20% NaCl solution, more than 22% survival is observed. Therefore, the P. acidilactici NRRL B-50517 freeze-dried products maintain the high resistance of Pediococcus probiotics to the high salt treatment.

TABLE-US-00011 TABLE 11 Detection of live probiotics from Pediococcus freeze-dried apples treated with different concentration of salts* Concentration of NaCl 30 minutes 1 hour 2 hours Overnight 0.1% 9.15 10.sup.7 1.03 10.sup.7 7.63 10.sup.6 3.13 01.sup.6 1.0% 3.63 10.sup.6 1.83 10.sup.7 1.73 10.sup.7 3.73 10.sup.6 5.0% 3.20 10.sup.6 1.81 10.sup.7 1.33 10.sup.7 3.05 10.sup.6 10.0% 1.09 10.sup.7 9.13 10.sup.6 6.13 10.sup.6 4.88 10.sup.5 20.0% 3.25 10.sup.6 9.28 10.sup.6 5.38 10.sup.6 7.13 10.sup.5 *Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the sliced apple into 5 ml different salt solutions (0.1%, 1.0%, 5.0%, 10.0%, and 20.0%) adjusted, mixed well, incubated at room temperature for thirty minutes, one and half hours and three hours. After incubation, transferred 100 l solution from the falcon tube, performed series of dilution, and plated 100 l of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies. Numbers were the average of two repeated experiments.

Example 12

Antibiotic Compatibility

[0046] Antibiotics are often prescribed to prevent and treat the infectious diseases. Adverse side effects of antibiotics are often reported, especially for those required to take the antibiotics for a long period. Probiotics are reported to decrease the side effects of antibiotics, especially related to digestive disorders. Pediococcus acidilactici is more tolerant to the antibiotics killing effects and is recommended as an adjunct to antibiotics treatment.

[0047] In order to determine whether freeze-drying interfered with this antibiotic compatibility, Pediococcus probiotic freeze-dried products were exposed amoxicillin at 10 g/ml, 50 g/ml, 100 g/ml, and 500 g/ml, and incubated at 45 C. for fours to see if any survival of Pediococcus acidilactici NRRL B-50517. Results summarized in table 12 indicate all of antibiotics treatment showed greater than 700 million live Pediococcus acidilactici NNRL B-50517 in one ml of MRS broth even at the high concentration of 500 g/ml of amoxicillin treatment. This result suggests that Pediococcus freeze-dried products can be applied together with antibiotics treatment as a substitute for Pediococcus acidilactici NRRL B-50517 fermentation powder products. For comparison, E. coli had 0% survival at 500 g/ml of amoxicillin (data not shown).

TABLE-US-00012 TABLE 12 Detection of live probiotics from Pediococcus freeze-dried apples treated with antibiotics for 4 hours* Concentration of Number of P. acidilactici NRRL Amoxicillin (g/ml) B-50517 (CFU/ml) 0 1.78 10.sup.9 10 7.38 10.sup.7 50 9.25 10.sup.7 100 7.13 10.sup.7 500 1.01 10.sup.8 *Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml MRS broth with 0 g/ml, 10 g/ml, 50 g/ml, 100 g/ml and 500 g/ml amoxicillin, incubated at 45 C. for 4 hours, and performed the detection of viability of P. acidilactici NRRL B-50517. After incubation, transferred 100 l solution from the falcon tube, performed series of dilution, and plated 100 l of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies. Numbers were the average of two repeated experiments.

[0048] After exposure probiotic freeze-dried products to elevated temperature, low pH, high salts, and antibiotics, reasonable amounts of live probiotics can still be detected from treated probiotic freeze-dried probiotics. These results indicate that probiotic freeze-dried products can be the alternative delivery of probiotics to humans and animals.

Example 13

Freeze-Dried Apples

[0049] In order to determine the amenability of various commercial strains of apples to serve as a substrate for delivery of freeze-dried bacteria a comparison was conducted. Results summarized in table 13 indicate that there is no significant difference between Red delicious, Fuji and Gala apples.

TABLE-US-00013 TABLE 13 Detection of live probiotics from Pediococcus probiotics freeze-dried apples* Numbers of P. acidilactici NRRL B- Variety of Apple 50517 (CFU/g)** Red Delicious 5.5 10.sup.8 Fuji 2.28 10.sup.8 Gala 2.18 10.sup.8 *Cut apples into 1/4 slices, then cut those into halves. Transferred apple slices into the Pediococcus acidilactici NRRL B-50517 solution [mixed 20 g of Pediococcus fermentative probiotics for freeze-dried powders (catalogue number PA-5051FD, Imagilin Technology, LLC) with 80 ml water], mixed the sliced apples and probiotic solution for 45 secs to a min, transferred Pediococcus coated apple on the freeze-drying trays covered with parchment paper and freeze-dried. **Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried apples, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies.

Example 14

Freeze-Dried Apples and Heat Treatment

[0050] In order to determine the effect of elevated temperature on probiotic bacteria freeze-dried onto foodstuffs, an experiment with different temperature conditions was conducted. Results, summarized in table 14 indicate that P. acidilactici NNRL B-50517 exhibited about 73% survival after 30 minutes at 65 C. and 30 minutes at 85 C.

TABLE-US-00014 TABLE 14 Survival of Probiotics after elevated temperature treatment of Pediococcus probiotic freeze-dried apples Number of P. acidilactici Treatment* NNRL B-50517 (CFU/g) % survival Room temperature 2.41 10.sup.8 100% 65 C., 30 min 2.32 10.sup.8 96.22% 65 C., 30 min followed by 2.35 10.sup.8 97.39% 85 C., 10 min 65 C., 30 min followed by 1.76 10.sup.8 72.92% 85 C., 30 min *Incubated Pediococcus probiotic freeze-dried apples at elevated temperature 65 C. for 30 minutes, 65 C. for 30-minute following by 85 C. for another 10 minutes or by 85 C. for another 30 minutes, then performed the detection of probiotics as described at Table 1. Control was to take the sliced Pediococcus freeze-dried apples at room temperature without being treated at elevated temperature and performed the probiotic assays directly.

Example 15

Freeze-Dried Sweet Potato and Heat Treatment

[0051] In order to confirm the results of example 14, an additional experiment was conducted using sweet potato as a substrate. Results, summarized in table 15 indicate that P. acidilactici NNRL B-50517 exhibited about 61% survival after 30 minutes at 65 C. and 30 minutes at 85 C.

TABLE-US-00015 TABLE 15 Survival of Probiotics after elevated temperature treatment of Pediococcus probiotic freeze-dried sweet potato Number of P. acidilactici NNRL B-50517 Treatment* (CFU/g) % survival Room temperature 4.32 10.sup.8 100% 65 C., 30 min 5.71 10.sup.8 132.40% 65 C., 30 min followed by 3.60 10.sup.8 83.41% 85 C., 10 min 65 C., 30 min followed by 2.64 10.sup.8 61.10% 85 C., 30 min *Incubated Pediococcus probiotic freeze-dried sweet potato at elevated temperature 65 C. for 30 minutes, 65 C. for 30-minute following by 85 C. for another 10 minutes or by 85 C. for another 30 minutes, then performed the detection of probiotics as described at Table 1. Control was to take the sliced Pediococcus freeze-dried apples at room temperature without being treated at elevated temperature and performed the probiotic assays directly.

Example 16

Freeze-Dried Apples Stored at 40 C.

[0052] In order to determine the effect of a 40 C. storage temperature on probiotic bacteria freeze-dried onto foodstuffs, an experiment was conducted using apples as a substrate. Results, summarized in table 16 suggest that P. acidilactici NNRL B-50517 is not adversely effected by exposure to 40 C. for up to 12 days.

TABLE-US-00016 TABLE 16 Detection of viable probiotics from Pediococcus probiotic freeze-dried probiotic apples stored at 40 C. Incubation Time for (days) at Numbers of Pediococcus % 40 C.* acidilactici NRRL B-50517 of survival control 3.25 10.sup.8 100% 4 1.63 10.sup.8 81% 7 5.00 10.sup.8 153% 12 4.14 10.sup.8 127% *Incubated Pediococcus probiotic freeze-dried apples at the elevated temperature 40 C. incubator. After 4 days, 7 days, and 12 days incubation, removed the elevated temperature treated Pediococcus probiotic freeze-dried apple, performed the detection of probiotics assays as described at Table 1. Control was to take the sliced Pediococcus freeze-dried apples at room temperature without being treated at elevated temperature and performed the probiotic assays directly.

Example 17

Freeze-Dried Apples Stored at 65 C.

[0053] In order to determine the effect of a 65 C. storage temperature on probiotic bacteria freeze-dried onto foodstuffs, an experiment was conducted using apples as a substrate. Results, summarized in table 17 suggest 1% to 2% of P. acidilactici NNRL B-50517 survive after 7 to 12 days.

TABLE-US-00017 TABLE 17 Detection of viable probiotics from Pediococcus probiotic freeze-dried probiotic apples stored at 65 C. Incubation Time for (days)at Numbers of Pediococcus % 65 C.* acidilactici NRRL B-50517 of survival control 3.25 10.sup.8 100% 4 2.65 10.sup.6 0.81% 7 6.38 10.sup.6 1.96% 12 3.88 10.sup.6 1.19% *Incubated Pediococcus probiotic freeze-dried apples at the elevated temperature 65 C. incubator. After 4 days, 7 days, and 12 days incubation, removed the elevated temperature treated Pediococcus probiotic freeze-dried apple, performed the detection of probiotics assays as described at Table 1. Control was to take the sliced Pediococcus freeze-dried apples at room temperature without being treated at elevated temperature and performed the probiotic assays directly.

Example 18

Viability of Bacteria on Various Substrates

[0054] In order to determine the amenability of various types of fruit and vegetables to serve as a substrate for delivery of freeze-dried bacteria a comparison was conducted. Results summarized in table 18 indicate that blueberry, strawberry, green bean and sweet potato are all suitable substrates for delivery of freeze-dried probiotic bacteria.

TABLE-US-00018 TABLE 18 Detection of live probiotics from Pediococcus freeze-dried food Numbers of P. acidilactici NRRL B- Type of Fruit or Vegetable 50517 (CFU/g)* Blueberry 7.33 10.sup.7 Green Beans 1.61 10.sup.8 Strawberry 1.93 10.sup.8 Sweet Potato 3.00 10.sup.8 *Measured 0.2 g to 0.4 g of Pediococcus probiotic freeze-dried blueberry, green berry, strawberry, or sweet potato, transferred the slice apple into 5 ml saline solution, and mixed well. Performed series of dilution, and plated 100 ul of desirable diluted solution onto MRS plates and incubated at the incubator overnight to count the colonies.

[0055] It should be understood that the claims provided herewith are not an exhaustive representation of the invention, but that further claims may be added.