Protection of microbial cells from acidic degradation

a technology of acidic degradation and microbial cells, applied in the direction of plant ingredients, food preparations, non-active ingredients of pharmaceuticals, etc., can solve the problems of reducing the effect of microbial cells, and reducing the formation of toxins, so as to enhance the weight gain of farm animals, enhance food utilisation, and improve the effect of mechanical strength

Inactive Publication Date: 2014-01-23
AUSTRIANOVA SINGAPORE PTE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0042]It is one embodiment of the invention to provide this technology for use in the food industry. Encapsulated microbial cells are especially useful to enhance weight gain in farm animals. Substitution e.g. of intrinsic microbial populations by microbial strains providing a more effective enzyme composition lead to an enhanced food utilisation. Therefore, the use of the technology and microcapsules as described above in the food industry is another embodiment of the invention.
[0043]By originating from a chemically defined starting material, surrounding the cells with such a cellulose based PEC capsule, which is not only of high mechanical strength and good biocompatibility, but also unaffected by acidic conditions, and able to respond to a change in the surrounding environment by releasing the cells and / or cell products from the capsules when passing through the intestine, a solution to the problem of the poor survival of the majority of cells, such as probiotics in dietary products and in orally administered food additives is achieved.

Problems solved by technology

A lack of proteases can cause incomplete digestion that can lead to allergies and the formation of toxins.
Simply swallowing additional amounts of digestive enzymes might not be the ideal solution, because the exposure to stomach acid when passing through the stomach might have a detrimental effect to the enzymes.
Additional problems in providing enzymes as a food supplement are the taste of such enzymes, some of them cause a “burning sensation” in the mouth, and their sensitivity towards moisture.
Non-encapsulated enzymes have been reported to lose their potency when they are exposed to normal air humidity, they therefore also cannot be taken as a drink or be taken as ingredient of a moist meal, unless added just before consumption.
This can lead to irritation.
Sometimes if enzymes are taken in a drink, which gets to the upper lip, proteases can linger and can cause a rash there.
Pancreatic enzymes are not stable at wide ranges in pH or temperature and are destroyed by stomach acid.
All these stresses result in death of a significant percentage of these cells.
This technique however may not be suitable for all food product applications because, firstly, the residual oil in the encapsulated material may be detrimental to texture and organoleptic characteristics, and may not be suitable for the development of low-fat dairy products.
Secondly, the residual oil, emulsifier and surfactant in the encapsulated material can be toxic to live microbial cells and may interact with sensitive food components.
However, the encapsulated microbial cells did not show a significantly increased survival rate when subjected to low pH and high bile salt conditions during in vitro tests.
It turned out that Lactobacillus acidophilus is more sensitive than Bifidobacterium, but that the encapsulation did not protect the microbial cells from being degraded by aqueous acidic solutions (Sultana et al.
The overall survival rates, however, were higher at pH 3.0 also for the encapsulated cells indicating that the effects of acidic stress cannot completely be prevented.
The big problem with this process is that it yields products with poor shelf-life (even when refrigerated) and poor survival in the stomach—all the CFU, literally 99.99%, get killed by stomach acid.”
While this system provides one solution to provide viable probiotics to customers, it is however not suitable for all uses as a food ingredient, as it is generating rather large capsules, which need to be swallowed intact, and may not be bitten open.

Method used

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  • Protection of microbial cells from acidic degradation
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  • Protection of microbial cells from acidic degradation

Examples

Experimental program
Comparison scheme
Effect test

example 1

Growing of Lactobacillus acidophilus to an OD of 1.0

[0102]A culture of Lactobacillus acidophilus was started with a 20 ul sample from the thawed bacteria stock by injecting it into 50 ml MRS (named by its inventors: de Man, Rogosa and Sharpe, developed in 1960; Preparation of 1 liter of MRS medium: 51 g MRS broth powder, 1 g Polysorbate 80, 0.5 g L-cysteine hydrochloride and 999 ml of H2O adjusted to pH of 6.2.) in a 50 ml EM flask. The stock had been kept at −80° C. and was purchased from DSM (catalogue number DSM 20079) (Moro) Hansen and Mocquot (ATCC 4356). The culture was incubated overnight shaking at 50 rpm and at 37° C. On Day 1 of the experiment, the optical density of the bacterial culture was determined at 600 nm on Tecan Infinite M200. Typically the optical density at 600 nm that gives a reading of 1 will correspond to the exponential phase of the bacterial growth. The cells were grown up to an OD 600 nm reading of 1, to ensure that cells were in the exponential phase bef...

example 2

Survival of Non-Encapsulated Lactobacillus acidophilus Cells in Hydrochloric Acid

[0103]A solution of 0.01M HCl in PBS (phosphate buffered saline) was prepared by adding 4.2 ml of 37% HCl to 500 ml PBS. The pH value was adjusted to 2.0 exactly by using 5M HCl.

[0104]5 ul of the lactobacillus culture was added to 1 ml of hydrochloric acid in PBS (phosphate buffered saline salt solution) in a sterile Eppendorf tube in triplicate. As a control, 5 ul of the same Lactobacillus culture was added to 1 ml of PBS in a sterile Eppendorf tube in triplicate at 0 hr time point. The hydrochloric acid testing was carried out at different time points, i.e. after 1 hr, 1.5 hr and 2 hrs of exposure time.

[0105]At the various time points all the Eppendorf tubes were centrifuged down at speed of 3000×g for 1 min to remove hydrochloric acid. They were washed twice with MRS medium and 100 ul of MRS medium was added into the pellet. The pellet was resuspended therein and all was transferred into a 96 well pl...

example 3

Encapsulation of Lactobacillus acidophilus Cells in NaCS and pDADMAC

[0107]100 ul of the bacteria culture with an optical density of 1 were mixed with 2 ml of sodium cellulose sulphate solution containing 1.8% sodium cellulose sulphate (09-5 ul-592, provided by the Fraunhofer Institute) and 1% sodium chloride, and dropped into a 150 ml bath of 1.3% 24 kDa pDADMAC with the use of a 5 ml syringe and a 23 G needle.

[0108]The hardening time for the capsules in the pDADMAC bath was 4 mins. The capsules were then washed once for 8 min with 300 ml of 1×PBS, and once 4 mins with 300 ml of 1×PBS. These were followed by 3 washes with 30 ml 1× Phosphate Buffered Saline each and 3 washes with 30 ml MRS medium each. The capsules were then transferred to a 250 ml conical flask containing 100 ml of fresh MRS medium. These capsules were cultured at 37° C. incubator, with a speed of 50 rpm.

[0109]The AlamarBlue Assay described above was performed on the encapsulated lactobacillus cells. The assay was p...

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Abstract

A simple cellulose sulphate based microencapsulation technology has been applied to encapsulate bacterial or other microbial cells, which produce and release digestive enzymes and thereby provides an acid resistant shelter for these microbial cells. Surprisingly, the resulting spheres were found to provide sufficient protection for encapsulated cells from treatment with aqueous acidic solutions. Thereby the cellulose sulphate microencapsulated cells, such as probiotics are now enabled to survive passage, for example, through the stomach after consumption by a human or animal with a higher survival rate than those not within a microcapsule. After passing the stomach these cells are delivering products produced by them, e.g. enzymes or other nutrition factors. This technology therefore proves to be very useful in providing digestive or otherwise beneficial enzymes and/or of living microbial cells, into the lower gastrointestinal tract, where they could confer their health benefit to the host.

Description

FIELD OF THE INVENTION[0001]The invention refers to a (simple) cellulose sulphate based microencapsulation technology which has been applied to encapsulate bacterial or other microbial cells which produce and release digestive enzymes and thereby provides an acid resistant shelter for these microbial cells. Surprisingly, the resulting spheres were found to provide sufficient protection for encapsulated cells from treatment with aqueous acidic solutions. Thereby the cellulose sulphate microencapsulated cells, such as probiotics are now enabled to survive passage, for example, through the stomach after consumption by a human or animal with a higher survival rate than those not within a microcapsule. After passing the stomach, these cells are delivering products produced by them, e.g. enzymes or other nutrition factors. This technology therefore proves to be very useful in providing digestive or otherwise beneficial enzymes and / or of living microbial cells, into the lower gastrointesti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/48A23L1/30A61K35/74A61K35/742A61K35/747
CPCA61K9/48A61K35/747A61K35/741A23L1/3014A23V2002/00A61K9/5026A61K9/5047A61K9/5031A61K35/742A61K36/064A23P10/30A23L29/06A23L29/262A23L33/135A61P1/04A61P1/12A61P19/02A61P3/00A61P3/04A23V2400/113A23V2200/224A23V2200/32A23V2250/51088A61K9/50A61K35/12
Inventor GUENZBURG, WALTER H.BRANDTNER, EVA MARIASALMONS, BRIAN SALMONSDANGERFIELD, JOHN A.
Owner AUSTRIANOVA SINGAPORE PTE
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