CZ307297B6 - The strain of Propionibacterium freudenreichii subspecies freudenreichii CCM 8774 and the use of its antifungal active metabolites in food and feed production - Google Patents

Abstract

The strain of Propionibacterium freudenreichii subspecies freudenreichii, deposited in the Czech Collection of Microorganisms of the Faculty of Science of Masaryk University, Kamenice 5, Brno under the collection number of CCM 8774. This strain is useful for producing propionic acid and a ferment containing antifungal active metabolites useful in the food and feed industry.

Description

The invention relates to the industrial microbial strain Propionibacterium freudenreichii subsp. freudenreichii CCM 8774, which is useful for the production of propionic acid and other antifungal active metabolites from lactic acid-containing substrates under industrial or continuous or discontinuous conditions.

BACKGROUND OF THE INVENTION

Most food and feed are susceptible to fiber fungal contamination at various stages of production, processing, transport and storage. Climatic and production, transport and storage conditions can promote fungal growth and the synthesis of mycotoxins leading to food spoilage or food safety. The trend in recent years in the production of food induced by consumer demands has been to reduce or eliminate the chemical compounds used for the preservation and protection of food and feed and to replace them with natural antimicrobial agents. It was the object of the present inventors to find ways to protect foodstuffs from spoilage caused by filamentous fungi by using bacteria producing antifungal metabolites.

SUMMARY OF THE INVENTION

These deficiencies are eliminated by a new industrial microbial strain of Propionibacterium freudenreichii subsp. freudenreichii CCM 8774 producing food grade propionic acid and acetic acid simultaneously with the production of other unspecified antifungal active metabolites when cultivated in lactic acid substrates.

Isolation of the strain according to the invention

To isolate the strain, 10 g of cheese was mixed with 90 ml of a 2% sodium citrate solution, and after making a homogeneous emulsion with the stomacher, decimal dilutions in saline were prepared and seeded on YEL agar (yeast extract lactate). Cultivation was anaerobic at 30 ° C for 6 days.

Characterization of the strain according to the invention

After purification of the colonies obtained, the isolates obtained were characterized by morphology and physiological features. Gram-positive and catalase-positive non-spore lactate-producing acetic acid and propionic acid bars were further characterized by genus-specific PCR, GTG5 primer-rep-PCR and 16S rRNA sequencing according to Rossi et al. in Syst. Appl. Microbiol. 21,419-28 of 1999. The genus-specific PCR has shown that the strain of the invention belongs to the genus Propionibacterium. Sequence analysis revealed 100% identity with the type strain of Propionibacterium freudenreichii subsp. freudenreichii. Fingerprint cluster analysis from repetitive PCR has also shown belonging to Propionibacterium freudenreichii subsp. freudenreichii.

The original strain according to the invention is deposited in the collection of dairy microorganisms Laktoflora under the number CCDM 1067 and in lyophilized state at the international deposit under the Budapest Treaty in the Czech Collection of Microorganisms of the Faculty of Science, Masaryk University, Kamenice 5, Brno.

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Determination of antifungal properties of the strain according to the invention

Preparation of extracts

Long-term frozen propionic acid bacteria strains were recovered in YEL broth (yeast extract lactate) at 30 ° C until growth and then inoculated (2% inoculum, v / v) into whey medium (acid whey retentate thickened by reverse osmosis, baseline pH) 4.4 to 4.5, lactic acid content (~ 2% w / v), whose pH was adjusted to 7.0 with NaOH prior to sterilization (110 ° C, 30 minutes). To maintain a constant pH during fermentation and as neutralizing agent, 5% (w / v) CaCO ₃ and 0.5% (w / v) trimagnesium phosphate (both components pre-sterilized at 121 ° C for 60 minutes) were added. After cultivation at 30 ° C, a portion of the fermented medium was sterile transferred to tubes and stored at -20 ° C until use (complete). The remaining portion was centrifuged at 9000 g for 10 minutes and the supernatant obtained was membrane filtered (0.2 µm) and also stored at -20 ° C (bb supematant).

Preparation of nutrient media and suspensions of filamentous fungi

The filamentous fungal strains used were cultured on barley at optimum temperature and after sporulation, the spores were extracted with saline containing 0.05% (w / v) Tween 80. The concentration of spores in the extract was determined using a Malt agar plate method (HiMedia Laboratories, Mumbai, India). The filamentous fungus spores were mixed into boiled broths at 50 ° C to a final concentration of 20 to 50 spores / ml medium and spilled onto the plates. The following nutrient media were used for testing:

Malt Agar M253 (HiMedia), pH 5.5 ± 0.2

Czapek Malt Agar M732 (HiMedia), pH 6.8 ± 0.2

JM3 agar: mixed 40 ml of concentrated whey RO pH 6.5 with 150 ml of deionized water and 10 g of barley flour, boiled to a homogeneous mixture, cooled to 25 to 28 ° C, pH adjusted again with 40% (m / v) NaOH to 6.5, sterilization 110 ° C, 20 minutes, sterile mixture mixed 12: 5 with boiled 3% agar.

Testing of antifungal properties

To test the efficacy of bb supernatants, mold spores in nutrient media were poured onto 18 cm diameter Petri dishes, and 8 mm diameter wells were punched in agar after solidification. 200 μΐ of the appropriate bb supernatant was pipetted into the wells and after 4 hours storage at 4-6 ° C, the plates were cultured at 25 ° C for 5 days. For live cell testing (complete), first 1.5 ml of the ensemble was pipetted onto the plate followed by 15 ml of spore suspension in the broth. Control plates included the addition of sterile acid whey pH 4.4-4.5 and neutralized whey pH adjusted to 7.0 with NaOH. The appearance of the plates was evaluated visually after 5 days by comparing the growth of the fungi on the plates with the test strain and controls containing acidic or neutralized whey (1 complete inhibition, 2 marked inhibition, 3 slight inhibition, 4 without inhibition) and photographic documentation was taken.

In the cell-free supernatants, only a slight inhibition of the growth of Penicillium nalgiovensis CCDM 329 was observed on both Malt and Czapek Malt agar by Propionibacterium freudenreichii subsp. freudenreichii CCM 8774. The same strain caused a significant inhibition of the growth of the Penicillium camemberti CCDM 767 and 769 test culture on Malt agar, whereas no inhibition was observed on Czapek Malt agar. In the case of Pen. roqueforti PA1, a significant growth inhibition was observed on this strain on Czapek Malt agar, while it was ineffective on Malt agar.

In the case of addition of live cells of propionic fermentation bacteria, the results are shown in Tab. 1.

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Table 1 Antifungal activity of living cells of various propionibacterial strains against filamentous fungi of the genus Penicillium

Similar results were obtained by Gwiazdowska et al. in Pol. J. Microbiol. 2008,57, 205-212, for Fusarium and Alternaria sponges where living cells were also more effective than cell-free supernatants.

For the production of propionic acid and ferment, this strain is used alone or in admixture with other strains, preferably for example with a strain of Lactobacillus rhamnosus VT1 and / or Kluyveromyces marxianus CCDM 269 in any ratio.

The following examples illustrate but do not limit the strain according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The starter propion culture maintained in the lyophilized state is recovered in 16% (w / v) skimmed milk with 0.5% (w / v) yeast extract and cultured at 30 ° C until the milk is clotted. The culture obtained is used to inoculate sterile whey (acid whey of 16-18% dry matter neutralized with NaOH to pH 6.5-7.0 prior to sterilization) with the addition of 2% sterile calcium carbonate. After 48 to 72 hours of static cultivation at 30 ° C, this culture is inoculated with a 25 L fermenter containing 20 kg of acidic microfiltered whey of 16-18% (w / v) dry matter containing about 2% (w / v) lactic acid in an amount of 5 to 10% inoculum. The whey during the fermentation is continuously neutralized to pH 6.5 to 7.0 with sodium hydroxide and is stirred (50 rpm). After two days of fermentation at 30 ° C, continuous fermentation is started and microfiltered whey of 14 to 18% dry matter and containing 4 to 10% (m / v) of sodium, calcium, ammonium lactate or mixtures thereof in the same volume as the fermented ferment is added. . During the fermentation, samples are taken continuously and the content of antifungal active substances is monitored. Fermentation is complete when a minimum content of 3% propionic acid (m / v) is reached.

Example 2

The fermentation is prepared as in Example 1, except that the addition of microfiltered whey having a dry matter content of 14 to 18% (w / v) and a content of 4 to 10% (w / v) of sodium, calcium, ammonium lactate or mixtures thereof is ferment after 24 to 48 hours of fermentation, inoculated with 0.01% (v / v) Lactobacillus rhamnosus VT1 bacterial culture capable of producing lactic acid from lactose contained in whey and the ferment is not continuously collected.

Example 3

The fermentation was prepared in the same manner as in Example 2, except that in conjunction with the propionic culture, it was inoculated with Kluyveromyces marxianus CCDM 269 at a dose of 1-5% (v / v).

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Example 4

The fermentation is prepared as in Examples 2 and 3 except that continuous neutralization is replaced by a single addition of max. 5% (w / v) calcium carbonate or a mixture of max. 5% (w / v) calcium carbonate and max. 5% (w / v) trimagnesium phosphate.

Example 5

The fermentation obtained according to Examples 1-4 is mixed with fermented whey containing 6-10% (w / v) lactic acid in a ratio such that the propionic acid content of the resulting mixture is greater than 3% (w / v) and at the same time the pH of the ferment was below 5.5.

Example 6

Microbial cells were removed from the ferment obtained in Example 4 by microfiltration using membranes having a porosity of 0.8 to 1.4 µm at a temperature of 25 to 50 ° C.

Example 7

The fermentation obtained in Examples 1 to 6 is concentrated on a vacuum evaporator at a temperature of 60 to 70 ° C to a dry matter of 20 to 50% by weight.

Example 8

The fermentation obtained according to Examples 1 to 6 is dried in a spray drier or by freeze-drying under these conditions of freezing -40 ° C, drying 0.4 mbar, drying 30 ° C, 0.02 mbar to a dry weight of at least 98% by weight.

Example 9

The ferment obtained according to Examples 5-8 is used as a substitute for the preservatives used in the toasted bread recipe. The fermentation rate is calculated so that after baking the propionate content of the bread does not exceed 3 g / kg. As a result of the use of the ferment, the shelf life of the toasted bread was prolonged by 4 to 6 days over the commercially used calcium propionate (Table 2).

Table 2: Comparison of the durability of toasted bread using different recipes (1)

PA content (%) m * (g) Water for dough (G) Yeast fresh (G) Dough temperature (’C) pH dough Yeasting (min.) Days to mold occurrence 0 control 0 0 810 41 30.7 5.84 60 5 6 6 6 5.75 calcium propionate 100 ALIGN! 6.3 810 50 31.0 5.86 60 12 13 13 14 13 Ferment MF 4 A 5.31 118 692 50 31.8 5.80 75 14 15 Dec > 21 > 21 17.75 Evaporator (tries) 7.69 82 728 50 31.1 5.88 75 15 Dec > 21 > 21 > 21 19.5

* m (g) = weight of preservatives into the dough

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Example 10

The fermentation obtained according to Examples 5 to 8 is used in the form of an aerosol for spraying onto the bread surface when hot, after being removed from the oven or after cooling.

Claims (6)

1. Industrial strain of Propionibacterium freudenreichii subsp. freudenreichii, deposited in the Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Kamenice 5, Brno under number 8774, which produces propionic acid and other antifungal active metabolites during whey cultivation. Use of the strain according to claim 1, for the production of a ferment in which the neutralized acid whey is fermented. Use of the strain according to claim 1, for the production of a ferment in which the neutralized acid whey is fermented and the fermentation is carried out with a mixture of at least two strains of microorganisms. Process for treating a ferment obtained according to claim 2 or 3, comprising concentrating the active compounds by concentration in a vacuum evaporator or by drying. A method of using the ferment obtained according to claims 2 to 4 as a substitute for preservatives in the dough for the preparation of toasted bread. A method of using the ferment obtained according to claims 2 to 4 as a substitute for preservatives by spraying onto the surface of bakery products. End of document