Technical Field

The invention is related to the production method of the microencapsulated form of probiotics with the chitosan-alginate polymers, the microcapsules loaded with probiotics obtained by this method and their use in food, agriculture and cosmetics.

Known State of the Art (Prior Art)

Mycotoxins are toxic metabolic products produced by some moulds such as Aspergillus, Penicillium and Fusarium, which can be found in foods and agricultural products generally as a result of contamination. Mycotoxins being formed during the cultivation, storage, processing or transportation of foods cause varying degrees of toxicosis (mycotoxicosis) in humans and/or animals. Preventing the growth of mould in foods is difficult, however, the amount of these can be minimized during the processing and storage of foods by providing hygienic conditions. Forming of moulds which produce toxins is an exceptional problem in the developing countries, that is because in these countries, there are no controlled storage conditions such as in the developed countries. Besides, in tropical zones having warm and humid climate, the risk of mould growth in food increases.

After the mycotoxin is formed, mycotoxins can be removed from the food products by binders. However, since this method is used after mycotoxin formation, it deteriorates the quality of the product, causes changes in taste and leads to chemical pollution. Also, since most of the binders used today are mycotoxin-specific, they do not present sufficient efficiency with products contaminated with more than one mycotoxin.

Due to reasons as such, the use of microorganisms which support consumer health and which have immune system-stimulating effects in the production of food and agricultural products increases. It is known that the probiotics which constitute a significant group of these microorganisms play an important role in the prevention and treatment of gastrointestinal system disorders and formation of normal m icroflora. Use of probiotics in food stuff is longstanding. However, addition of probiotics directly into the product causes change in taste and quality.

Also, it is known that the probiotic technology boosts the imm une system , repairs the natural defence mechanism , prevents collagen structure damage and slows the aging process by providing the water balance of the skin .

When the inventions similar to the subj ect invention are analysed, the following documents are found :

• US2006000851 1 A1 : l n this patent document, a preparation , which comprises a probiotic encapsulated with a m ix of xanthan gum and chitosan gum as ingredients, developed for pets, is described.

• US20120128821 A1 : l n this patent docu ment, a method related to isolating probiotic from donkey’s milk is described.

Brief Description and Purposes of the I nvention

The invention is related to microcapsules obtained by m icroencapsulating endospore form of probiotics with chitosan-alginate polymers. The m icrocapsules loaded with said probiotics prevent mycotoxigenic moulds and the mycotoxins which the moulds produce through biological struggle. I n this study, encapsulation of probiotics is carried out especially in order to prevent probiotics from being damaged by gastric acid or in order to increase the quality in fermented products.

The microcapsules loaded with probiotics related to the subj ect invention ;

Prevent the contamination of mycotoxin to food products at the rate of 95% with different combinations of probiotics,

Do not cause any alteration in the colour and appearance of the product they are applied to,

- Affect many mycotoxin species of different types since they suppress the mould growth and stress mechanism in food and agricultural products, Have antifungal effect,

Do not necessitate extra disinfection since at the same time, they will also have an antifungal effect on the product they are being applied to,

- Are natural and harmless,

Provide protection in storage up to 360 days by extending the shelf life of the product they are applied to.

Detailed Description of the I nvention

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.

The production method of the microcapsules loaded with the probiotic related to the invention contains the procedure steps of ;

Dissolving the alginate in water which is between 0,002- 0,1 % ratio by weight,

- Autoclaving the alginate solution at 1 10-130C°temperature, preferably at 121 C ⁰ and then sterilizing the same,

Mixing the probiotics with alginate,

- Adding calcium chloride (CaCI ₂ ) between 0,01 -1 M, preferably 0,1 M to the probiotic- alginate mix and stirring,

Dissolving chitosan in 0,25-3% 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 (I n this stage, the pH is adj usted between 3.5-7) .

- Autoclaving the chitosan solution at 1 10-130°C temperature, preferably at 121 °C and then sterilizing the same,

- Thereafter, combining the 3: 1 chitosan solution and the alginate-calcium chloride (CaCI ₂ ) solution containing the probiotics in a container dropwise,

Adding tween 80 (Polysorbate 80) between 0.01 -2% by volume and stirring the mixture until homogenization is obtained. The probiotics used in the invention are chosen from the microorganisms of ; Bacillus laterosporus, Bacillus sphaericus, Bacillus subtiHs, Bacillus coagulans, Streptococcus thermophilis, Azotobacter, Azospirillum, Agrobacterium, Gluconobacter, Havobacterium, HerbaspiriHum, Bacillus megaterium, ThiobaciHus, , B. polymyxa, B. brevis, B. Hcheniformis, B. circulans, B. cereus, B. thuringiensis, B. longum, B. breve, B. infantis, L. he/veticus, L. rhamnosus, L. plantarum, L casei , L. acidophilus , Lactobacillus delbreckii, Lactobacillus ellobiosus, Lactobacillus lactis, Lactobacillu cidophi/us, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus he/veticus, Streptococcus cremoris, Streptococcus thermophiles, Streptococcus intemedius, Streptococcus lactis, Streptococcus diacetHactis, 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 iongum, Leuconostoc spp. L. saiivarius, L. paracasei, L. gasseri, L. reuteri, B. Bifidum, B. iongum, B. infantis, Lb. deibrueckii, Lb. plantarum, Lb. pentosus, Lb. brevis, P. damnosus, Lb. coiiinoides, Lb. pentosus, Pediococcus spp., Lb. buohneri, Leuconostoc mesenteroides, Pedococcus pentosaceus, Lb. casei, Lb. kefir, Lb. acidophilus, Lb. he/veticus, Lb. casei, Lb. buigaricus, Lb. iactis, Lb. plantarum, Lb. brevis , Acetobacter spp., Streptococcus genera, Streptococcus spp., Streptococcus iactis, S. thermophiius, S. durans, S. cremoris, Lactobacillus aiimentarus, L. Aiimentarus, L. muitaromicus, L. sanfrancisco, Lactococcus iactis ssp, S. cerevisia, Lactobacillus sakei, Lactobacillus aiimentarius, Lactobacillus paraiimentarius, Lactobacillus paracasei, Lactobacillus buchneri, Enterococcus faecium, Enterococcus mundtii, Enterococcus faeceiis, Enterococcus casseiifiavus, Lactobacillus pentosus, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillus farciminis, Pi chi a kudriavzevii, Lactobacillus farciminis, Lactobacillus casei, Lactobacillus aiimentarius, Pichia kudriavzevii , Candida humi/is, L. iactis subsp. cremoris, Lb. deibrueckii subsp. iactis, Lb. he/veticus, Lb. casei, Lb. deibrueckii subsp. , Leuc. mesenteroides subsp. cremoris, Lb. johsonii, Lb.kefirenofacies, Lb. curvatus, P. acidilactici, P.pentosaceus, Lb. aiimentarius, C.piscicoia, Leuc. mesenteroides, Pacidiiactici, P. cerevisiae, Lb. pentosus, Pacidiiactici, T.haiophiius, Lb.sanfransiscensis, Lb. farciminis, Lb. fermentum, Lb.amyiovorus, Lb. reuteri, Lb.pontis, Lb.panis, Lb. aiimentarius, W.cibaria, O.oeni, L.coryniformis, L. curvatus, L.jugurti, L.jensenii, L.bucheneri, L.ce/iobiosus, L.coprophiius, L.hiigardii, L.ieichmannii, Ldextranicum, Pacidiiactici, P.pentosaceus, S. thermophiius, L. iactis subsp. diacety iactis, L/actis subsp. hordniae, Lgarvieae, L.rafinoiactis, VJluvialis, V.salmoninarum, Leuconostoc sp., Lcremoris, Ldextranicum, Lmesenteroides, L.paramesenteroides, L.gelidum, Lcarnosum, Carnobacterium sp., C.divergens, C. mobile, GgaHinarum, C.piscicola, Vagococcus sp., VJluvialis, V.salmoninarum, L.garvieae, Lactococcus diacetylactis, Propionibacterium freudenreichii, Pediococcus sp, S.uvarum, Lb.coryniformis, Candida crusei, Weisseiia confusa, Hansenuia siivicoia, Debaryomyces hansenii, Trichosporon beigeiii, Bacillus amyloliquefaciens, Toruiopsis sp., Candida my coderm a, Lb.buchneri, Pacidiiactici, Pediococcus pentasaceus, L.ceiiobiosus, Emundtii/Egaiiinarum, Ecasseiifiavus, Purinae-equi, Lb.murinus, Candida milled, E.burtonii, EJibu linger, issatchenkia oriental is, Candida peiiicuiosa, C. tropical is, Pediococcus acid ii act ici, Pseudopiantarum, Pediococcus acidiiactici, Pediococcus pentosaceus, Leuconostoc pseudomesenteroides, Weisseiia cibaria, Lb. parapiantarum, Issatchenkia orientaiis, Candida giabrata, Pediococcus acidiiactici, Kiuyveromyces marxianus, Pi chi a kudriavzevii , Saccharomyces servazzi, Toruiaspora deibrueckii, Kazachstania unispora, Saccharomyces bar nett ii.

The application of the product related to the invention to food and agricultural products is as follows:

10 ml of the solution containing 10 ⁹ cfu bacteria is mixed with 1 L water in order to provide protection against mycotoxin in the garden or during the storage period, - The same is applied on the products by means of a spraying method.