Microcapsules loaded with probiotics and production thereof

Abstract

A production method of microencapsulated probiotics with chitosan-alginate polymers, the microcapsules loaded with probiotics obtained by this method and their use in food, agriculture and cosmetics. The microcapsules do not make alterations in the color and appearance of the product in which they are applied and have antifungal and anti-mycotoxin features.

Claims

1. A method of producing polymeric microcapsules containing a probiotic which are protective against mold and mycotoxin formation, comprising the steps of: 1) dissolving alginate in water at a ratio by weight in a range of 0.002-0.1% to obtain an alginate solution, 2) autoclaving the alginate solution at a temperature range of 110-130 degrees C. and then sterilizing the alginate solution, 3) mixing a probiotic with the alginate solution to obtain a probiotic-alginate solution, 4) adding calcium chloride (CaCl.sub.2) in a range of 0.01-1.0 M to the probiotic-alginate solution and stirring to obtain an alginate-calcium chloride solution, 5) dissolving chitosan in a 0.25-3.0% concentration range by weight in 0.5-5% organic acid by volume to obtain a chitosan solution, 6) autoclaving the chitosan solution at a temperature range of 110-130 degrees C., and then sterilizing the chitosan solution, 7) combining dropwise in a ratio of 3:1 the chitosan solution and the alginate-calcium chloride solution containing the probiotics in a container to obtain a chitosan-alginate-calcium chloride mixture, 8) adding Polysorbate 80 to the chitosan-alginate-calcium chloride mixture in a range of 0.01-2% by volume and stirring the mix until homogenization is obtained.

2. The method of claim 1, wherein the organic acid comprises at least one of lactic acid, acetic acid, ascorbic acid, citric acid, tartaric acid and malic acid.

3. The method of claim 1, wherein pH is in the range of 3.5-7 when the chitosan is dissolved in the organic acid.

4. The method of claim 1, wherein the alginate solution is autoclaved at a temperature of 121 degrees C. and then sterilized.

5. The method of claim 1, wherein the chitosan solution is autoclaved at a temperature of 121 degrees C. and then sterilized.

6. The method of claim 1, wherein 0.1 M calcium chloride (CaCl.sub.2) is added to the probiotic-alginate solution and stirred.

7. The method of claim 1, wherein the chitosan is dissolved in 1% organic acid.

8. The method of claim 1, wherein the probiotics comprise at least one of bacteria and yeast.

9. The method of claim 8, wherein the probiotics are microorganisms selected from the group consisting of Acetobacter spp., Agrobacterium, Azospirillum, Azotobacter, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus laterosporus, Bacillus megaterium, Bacillus sphaericus, Bacillus subtilis, Bifidobacterium spp., Bifidobacterium adolecentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Candida glabrata, Candida humilis, Candida krusei, Candida milleri, Candida mycoderma, Candida pelliculosa, Carnobacterium sp., Debaryomyces hansenii, Enterococcus casseliflavus, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus mundtii, Gluconobacter Flavobacterium, Hansenula silvicola, Herbaspirillum, Issatchenkia orientalis, Kazachstania unispora, Kluyveromyces marxianus, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus paralimentarius, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Lactococcus spp., Lactococcus diacetylactis, Lactococcus lactis ssp., Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. diacetylactis, Lactococcus lactis subsp. hordniae, Leuconostoc spp., Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides, Oenococcus oeni, Pediococcus spp., Pediococcus acidilactici, Pediococcus cerevisiae, Pediococcus pentosaceus, Pediococcus urinae-equi, Pichia kudriavzevii, Propionibacterium freudenreichii, Pseudoplantarum, Saccharomyces barnettii, Saccharomyces servazzi, Streptococcus spp., Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus intermedius, Streptococcus lactis, Streptococcus thermophilus, Thiobacillus, Torulaspora delbrueckii, Torulopsis sp., Trichosporon beigelli, Vagococcus spp., Vagococcus fluvialis, Vagococcus salmoninarum, Weissella cibaria, Weissella confusa.

10. The method of claim 1, wherein the probiotic comprises an endospore.

Description

DETAILED DESCRIPTION

(1) The present invention is related to chitosan-alginate microcapsules loaded with probiotics which present antifungal and anti-mycotoxin activity in fields such as food, agriculture, cosmetics and healthcare.

(2) The production method of the microcapsules loaded with probiotic related to the invention contains the following steps of;

(3) Dissolving alginate in water at a ratio of 0.002-0.1% by weight, Autoclaving the alginate solution at a temperature of 110-130 degrees C., preferably at a temperature of 121 degrees C. and then sterilizing the same, Mixing probiotics with the alginate solution, Adding calcium chloride (CaCl.sub.2) in an amount between 0.01-1.0 M, preferably 0.1 M to the probiotic-alginate mix and stirring, Dissolving chitosan in a 0.25-3.0% concentration by weight in 0.5-5% organic acid by volume (lactic acid, acetic acid, ascorbic acid, citric acid, tartaric acid, malic acid etc.), preferably in 1% organic acid (In this stage, the pH is adjusted between 3.5-7). Autoclaving the chitosan solution at a temperature of 110-130 degrees C., preferably at 121 degrees C. and then sterilizing the same, Thereafter, combining the 3:1 chitosan solution and the alginate-calcium chloride (CaCl.sub.2) solution containing the probiotics in a container dropwise, Adding tween 80 (Polysorbate 80) to the chitosan solution-alginate-calcium chloride mix in a range of 0.01-2% by volume and stirring the mixture until homogenization is obtained.

(4) The probiotics used in the invention are chosen from the microorganisms of; Bacillus laterosporus, Bacillus sphaericus, Bacillus subtilis, Bacillus coagulans, Streptococcus thermophilis, Azotobacter, Azospirillum, Agrobacterium, Gluconobacter, Flavobacterium, Herbaspirillum, Bacillus megaterium, Thiobacillus, B. polymyxa, B. brevis, B. licheniformis, B. circulans, B. cereus, B. thuringiensis, B. longum, B. breve, B. infantis, L. helveticus, L. rhamnosus, L. plantarum, L. casei, L. acidophilus, Lactobacillus delbreckii, Lactobacillus ellobiosus, Lactobacillus lactis, Lactobacillu cidophilus, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus helveticus, Streptococcus cremoris, Streptococcus thermophiles, Streptococcus intemedius, Streptococcus lactis, Streptococcus diacetilactis, Enterococcus feacalis, Lactococcus spp, Lactococcus lactis subsp., Pediococcus spp., Pediococcus cerevisiae, Pediococcus acidilactici, Pediococcus pentosaceus, Bifidobacterium spp. Bifidobacterium animalis, Bifidobacterium adolecentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Leuconostoc spp. L. salivarius, L. paracasei, L. gasseri, L. reuteri, B. Bifidum, B. longum, B. infantis, Lb. delbrueckii, Lb. plantarum, Lb. pentosus, Lb. brevis, P. damnosus, Lb. collinoides, Lb. pentosus, Pediococcus spp., Lb. buohneri, Leuconostoc mesenteroides, Pedococcus pentosaceus, Lb. casei, Lb. kefir, Lb. acidophilus, Lb. helveticus, Lb. casei, Lb. bulgaricus, Lb. lactis, Lb. plantarum, Lb. brevis, Acetobacter spp., Streptococcus genera, Streptococcus spp., Streptococcus lactis, S. thermophilus, S. durans, S. cremoris, Lactobacillus alimentarus, L. Alimentarus, L. multaromicus, L. sanfrancisco, Lactococcus lactis ssp, S. cerevisia, Lactobacillus sakei, Lactobacillus alimentarius, Lactobacillus paralimentarius, Lactobacillus paracasei, Lactobacillus buchneri, Enterococcus faecium, Enterococcus mundtii, Enterococcus faecelis, Enterococcus casseliflavus, Lactobacillus pentosus, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillus farciminis, Pichia kudriavzevii, Lactobacillus farciminis, Lactobacillus casei, Lactobacillus alimentarius, Pichia kudriavzevii, Candida humilis, L. lactis subsp. cremoris, Lb. delbrueckii subsp. lactis, Lb. helveticus, Lb. casei, Lb. delbrueckii subsp., Leuc. mesenteroides subsp. cremoris, Lb. Joshin, Lb. kefirenofacies, Lb. curvatus, P. acidilactici, P. pentosaceus, Lb. alimentarius, C. piscicola, Leuc. mesenteroides, P. acidilactici, P. cerevisiae, Lb. pentosus, P. acidilactici, T. halophilus, Lb. sanfransiscensis, Lb. farciminis, Lb. fermentum, Lb. amylovorus, Lb. reuteri, Lb. pontis, Lb. panis, Lb. alimentarius, W. cibaria, O. oeni, L. coryniformis, L. curvatus, L. jugurti, L. jensenii, L. bucheneri, L. cellobiosus, L. coprophilus, L. hilgardii, L. leichmannii, L. dextranicum, P. acidilactici, P. pentosaceus, S. thermophilus, L. lactis subsp. diacety lactis, L. lactis subsp. hordniae, L. garvieae, L. rafinolactis, V. fluvialis, V. salmoninarum, Leuconostoc sp., L. cremoris, L. dextranicum, L. mesenteroides, L. paramesenteroides, L. gelidum, L. carnosum, Carnobacterium sp., C. divergens, C. mobile, C. gallinarum, C. piscicola, Vagococcus sp., V. fluvialis, V. salmoninarum, L. garvieae, Lactococcus diacetylactis, Propionibacterium freudenreichii, Pediococcus sp, S. uvarum, Lb. coryniformis, Candida crusei, Weissella confusa, Hansenula silvicola, Debaryomyces hansenii, Trichosporon beigelli, Bacillus amyloliquefaciens, Torulopsis sp., Candida mycoderma, Lb. buchneri, P. acidilactici, Pediococcus pentasaceus, L. cellobiosus, E. mundtii/E. gallinarum, E. casseliflavus, P. urinae-equi, Lb. murinus, Candida milleri, E. burtonii, E. fibulinger, Issatchenkia orientalis, Candida pelliculosa, C. tropicalis, Pediococcus acidilactici, Pseudoplantarum, Pediococcus acidilactici, Pediococcus pentosaceus, Leuconostoc pseudomesenteroides, Weissella cibaria, Lb. paraplantarum, Issatchenkia orientalis, Candida glabrata, Pediococcus acidilactici, Kluyveromyces marxianus, Pichia kudriavzevii, Saccharomyces servazzi, Torulaspora delbrueckii, Kazachstania unispora, Saccharomyces barnettii.

(5) Exemplary application of the product related to the invention to food and agricultural products is as follows: 10 ml of the solution containing 10.sup.9 cfu bacteria is mixed with 1 L water in order to provide protection against mycotoxin in the garden or during the storage period.

(6) The same is applied on the products by means of a spraying method.