WO2017216818A1

WO2017216818A1 - Anaerobic growth of bacteria in unicellular alga

Info

Publication number: WO2017216818A1
Application number: PCT/IT2016/000158
Authority: WO
Inventors: Beatrice LETER; Raffaella BRUSCHI
Original assignee: Ocean Farma Srl
Priority date: 2016-06-17
Filing date: 2016-06-17
Publication date: 2017-12-21

Abstract

Method of culture of an anaerobic bacterium, obligate or facultative, in particular Lactobacillus sp., which comprises the inoculation of the anaerobic bacteria in an extract, homogenized or entire, of the unicellular microalgae in a solvent medium under anaerobic conditions. The unicellular microalgae have a concentration in the solvent medium able to sustain the growth of the anaerobic bacteria. With this method, a fermented comprising both a bacterial culture medium and a bacterial population grown in it is obtainable. The fermented is suitable to be inserted into the composition of a constituent source of a bacterium, in particular a probiotic, and, at the same time, of a fermented, in particular rich in omega - 3, for the growth of it, for nutritional, therapeutic and cosmetic use; in particular it can be used as a food supplement.

Description

ANAEROBIC GROWTH OF BACTERIA IN UNICELLULAR ALGA The present invention relates to a novel method for microbiological culture. More particularly, the present invention relates to a method of culture of a probiotic anaerobic bacterium, both obligate and facultative in unicellular algae, with a high nutritional value for human supply, in particular rich in omega-3. Nowadays probiotics based on different bacterial strains are available on the market (generally Lactobacilli or Bifidobacteria) . These probiotics produce lactic acid by fermentation of sugars, in different formulations and concentrations. The beneficial properties of lactic acid are widely recognized, some closely related with bowel health, through the edit action of the intestinal flora.

The positive effects of fermented foods (e.g. Yogurt, cheese) on the human organism have been known for a long time. Moreover, the nutritional qualities of different unicellular algae are also known (eg. Spirulina, Chlorella, Dunaniella) . These algae are rich in protein, minerals and trace elements and they are already greatly appreciated as supplements in the human diet.

Some of these algae are particularly rich in polyunsaturated fatty acids called omega-3, which make them particularly interesting, as the 00158 beneficial and curative properties of omega-3 are scientifically proven.

However, despite the known health benefits of probiotics and omega-3, nowadays, a product containing probiotic bacteria grown in unicellular algae rich in omega-3 doesn't exist on the market.

The present invention aims to provide a product that combines the beneficial properties of probiotics and the ones of unicellular algae rich in omega-3 fatty acids. Specifically, the present invention arises the purpose of providing a product based on probiotic strains grown in unicellular algae rich in omega - 3 which combines the beneficial properties of the cultivated probiotics with the omega - 3 fatty acids .

More in general, the present invention aims to provide a product suitable to constitute the source of probiotic bacteria grown under anaerobic conditions (fermentation) in a culture medium, based on unicellular algae rich in omega - 3, which is suitable for the growing of bacterial strains and, at the end of the bacterial culture, it must not be removed because it provides an important contribution to the human diet, as a source of omega - 3.

It should be emphasized that this preparation has nutritional value (since the probiotic, during the fermentation time, consumes the glucose component of a unicellular alga, leaving unchanged the lipid portion rich in omega-3 available to the human consumption) and therapeutic (both the curative effects of the probiotic and the anti inflammatory and anti - oxidant of the omega - 3 are known) and cosmetic properties (different probiotic strains produce metabolites of glycerin and other moisturizing , ions and heavy metals, where ^Λ bacterial metabolite ' means any molecule of neo-synthesis that is metabolized by a bacterium, which is economically interesting and obtained by anaerobic growth) .

The Inventors have found that an homogenized unicellular microalgae, either encapsulated or free of the capsule, is accessible from a bacterium, which is able to grow inside it in anaerobic conditions. Thus, the aforementioned objectives are achieved with a method of culture of an anaerobic bacterium both obligate or facultative, which is inoculated in a homogenised or entire extract of unicellular microalgae in a solvent under anaerobic conditions; the unicellular microalgae has a concentration in the solvent able to sustain the growth of the anaerobic bacteria. The present invention possibly involves the further addition of a complement of sugar to the extract of unicellular microalgae in the method of culture of the anaerobic bacteria. The method presented in this invention is economically advantageous and useful for the activation of the metabolism of the anaerobic bacterium and for its tropism in the intestinal lumen.

Moreover, the method makes superfluous each subsequent purification step of the bacterium from its growth medium, which in turn has an important nutritional value in the lipid content and therapeutic properties due to the presence of omega - 3.

Differently to what has been done so far in the use of unicellular algae with industrial purposes, for example as biofuels, or cosmetic, the approach of the present invention does not contain any extraction of the organic phase or enrichment of the lipid phase. The microalga is used in its entirety, since the component of sugar and protein is functional to the growth of the bacterium, while the lipid portion is maintained as a source of omega - 3 fatty acids in the final fermented product .

As explained above, in the present invention it is contemplated that, for the method of culture of an anaerobic probiotic bacterium, the extract of single-cell microalgae is an extract of Nannochloropsis gaditana homogenized and, in particular, that the extract of Nannochloropsis gaditana has been obtained starting from a fresh culture of Nannochloropsis gaditana in the exponential phase of growth.

It is also contemplated that in the method of culture of an anaerobic bacterium according to the present invention, as described above, the single- cell microalgae extract is an extract of Isochrysis sp. And, more particularly, that the extract of Isochrysis sp. has been obtained from Isochrysis sp. lyophilized. A product containing a fermented is so obtained, comprising in combination both a bacterial culture medium and a bacterial population grown in it, obtained by the method of culture of an anaerobic bacterium as described above, for which the product is suitable as source of probiotic and simultaneously as source of a fermented of the unicellular alga containing omega- 3 for nutritional, therapeutic or cosmetic use.

Moreover, in the present invention it is contemplated that, in the method of culture of an anaerobic bacterium, as described above, Lactobacillus sp. is the inoculated anaerobic bacterium. A product containing an entire fermented is so obtained as described above, wherein the anaerobic bacterium is Lactobacillus sp., for which the product is suitable as a source of probiotic and, at the same time, as a dietary supplement of the fermented of algae, rich in omega - 3, used for the growth of the bacterium. Thus, the present invention defines a culture technique of obligate or facultative anaerobic bacteria, which does not resort to the microbiological culture media available on the market, and uses an entire or homogenate extract of single-cell microalgae in oxygen-free conditions as the sole source of proliferation of the bacteria.

The fermented product obtained with the method of the present invention is completely free of animal elements, respect to probiotics grown in classical culture broths, containing extracts of lean meat, rich in amino acids, peptides, nucleotides, organic acids, vitamins and minerals. Therefore, a method of culture of an anaerobic bacterium, obligate or facultative, as recited in Claim 1 attached hereto, constitutes the object of the present invention.

In addition, object of the present invention is also a method of culture of an anaerobic bacterium, obligate or facultative, as recited in Claim 2 attached hereto. Preferred examples of this method are recited in the dependent claims 3; 4; 5 ; 6, e 7. Object of the present invention is also a product containing an entire fermented, comprising in combination both a medium of bacterial culture and a bacterial population grown in it, as recited in the attached claim 8. A preferred example of this method is recited in the attached dependent claims 9. The present invention will be fully understood based on the following detailed description of the best way of implementing it, given only as example, absolutely not limitative of the invention itself, in reference to the attached drawings, in which:

- FIG 1 is a diagram which shows the curves of bacterial growth under anaerobic conditions in an extract of a first strain of the unicellular alga in a bacterial culture according to the method of the present invention and in a culture media commercially available, and

- FIGURE 2 is a diagram showing the curves of bacterial growth under anaerobic conditions in an extract of a second strain of the unicellular alga in a bacterial culture according to the method of the present invention and in a culture medium commercially available.

The present invention is a method of culture of an anaerobic bacterium, obligate or facultative, that inoculates anaerobic bacteria in a homogenate or entire extract of the unicellular alga in a solvent under anaerobic conditions. The concentration of the unicellular alga in the solvent has to be effective to sustain the growth of anaerobic bacteria.

It is contemplated to add a complement of sugars to the extract of single-celled algae.

EXAMPLE I

In this Example I, the extract of unicellular algae is an homogenized extract of Nannochloropsis gaditana and the anaerobic bacterium, from the strain Lactobacillus sp..

Nannochloropsis gaditana is a spheroidal microalgae of diameter about 2 - 3 micrometers, devoid of motor structures. Its lipid content is about 30% of its dry weight. It accumulates large concentrations of various pigments, such as, for example, astaxanthin, canthaxanthin and zeaxanthin. Nannochloropsis gaditana is also a food product and, indeed, it is used to produce dietary- supplements. It is also served at the restaurant in Spain in El Puerto de Santa Maria in Cadiz, in the area where it was isolated for the first time and where it still grows wild. For Nannochloropsis gaditana the bioavailable sugars were measured in the laboratory by the Inventors and result to be dependent on the lysis method of microalgae and constitute from 4 % to 8 % of microalgae dry mass, distributed as follows: from 40% to 50 % of glucose; from 18% to 23 % of galactose; from 13% to from 2% to 4 % of xylose ; from 3% to 5 % of ribose ; from 4 % to 8 % other sugars .

In the present example the extract Nannochloropsis gaditana may have been obtained from a fresh culture of Nannochloropsis gaditana in exponential phase of growth, and the micro-algae of the strain of Nannochloropsis gaditana was subjected to a mechanical homogenization process via a probe homogenizer with teflon head, by homogenization of 2 minutes at 14,000 rounds per minute. Since the purpose of the extract of mxcroalga was to sustain the growth of the bacteria in the anaerobic fermentation conditions, the parameter for the choice of the concentration of microalgae in physiological saline solution was determined based on obtaining an amount of sugar comparable with that described in the literature as sufficient to maintain fermentative metabolism of Lactobacillus sp., that is 10 grams per liter. In order to not compromise the isotonicity of the culture medium, the amount of 100 grams of dry mass per liter of physiological solution it was never exceeded.

The concentration of extract of Nannochloropsis gaditana was less than 10 grams per litre. With this dilution the Inventors verified the growth with the extract of algae, but also with an addition of a complement of 5 grams of glucose per liter and with a culture medium MRS DE MAN - ROGOSA - SHARPE for lactobacilli , commercially available. In the exposed experiment a sterility control of the culture medium and of the extract of microalgae was included. Under the experimental conditions of Example I, the growth took place into sealed tubes, in fact, any induced correction of the acidity produced by lactic acid fermentation was not expected, it was therefore expected that the fermentation was stopped because of the acidification of the culture medium and because of sugar depletion.

The results of the growth of Lactobacillus sp. in the extract of Nannochloropsis gaditana, fresh and homogenized, are illustrated in FIGURE 1, in which: the curve 1 is the growth curve obtained with the commercially available MRS culture medium; the curve 2 is the growth curve of Lactobacillus sp. in the extract of Nannochloropsis gaditana (homogenized) ; curve 3 is the growth curve of Lactobacillus sp. in the extract of Nannochloropsis gaditana (homogenized) with the addition of 5 grams of glucose per liter of physiological solution. Surprisingly the inventors revealed that, in the aforementioned experimental conditions, the suitably homogenized extract of Nannochloropsis gaditana was able to support the growth of Lactobacillus sp. without the aid of any sugar integration, even if an addition of sugar sensibly improves the yield, at least for three days starting from the inoculation of the bacterium.

EXAMPLE II

In the Example II the extract of unicellular microalgae is an extract of Isochrysis sp. and the strain of the anaerobic bacterium is Lactobacillus sp.. Isochrysis sp. is a brown and flagellated unicellular alga, about 5 micrometers long and widely used in aquaculture. It has a high content of docosahexaenoic acid or DHA (omega - 3 fatty acid is the most effectively under the curative profile) and Isochrysis sp. is often used to enrich zooplankton (rotifers or Artemia) . It is considered a primary algae and it is the most commonly used to raise oysters, clams, mussels, scallops and in the shellfish farms. The high content of omega3 and sugar (about 30% of the dry weight) of Isochrysis sp., in addition to the absence of a cellulose capsule, makes Isochrysis sp. particularly suitable for the purposes of the present invention. The advantage of Isochrysis sp. is to have a high content of sugars and not to possess a cellulose capsule. This latter feature ensures that it is accessible to a bacterium and that is able to ensure the growth also in a non-homogenized state. On the contrary an encapsulated microalga is of difficult access for a bacterium and therefore the culture is not possible without prior homogenization. In this Example II the extract of Isochrysis sp. was obtained from Isochrysis sp. lyophilized. Even under the experimental conditions of Example II the growth took place in sealed tubes in such a way that the application of any induced correction of acidity produced by lactic acid fermentation was not expected. It was therefore expected that the fermentation was stopped by the acidification of the culture medium and cause of sugar depletion.

The results of the growth of Lactobacillus sp. in Isochrysis sp . extract are illustrated in FIGURE 2, in which: the curve 11 is the growth curve obtained with the commercially available MRS culture medium; the curve 12 is the growth curve of Lactobacillus sp. in the entire extract of Isochrysis sp.; the curve 13 is the growth curve of Lactobacillus sp. in the entire extract of Isochrysis sp., with the addition of 5 grams of glucose per litre of physiological solution; the curve 14 is the growth curve of Lactobacillus sp. in the homogenized extract of Isochrysis sp.; the curve 15 is the growth curve of Lactobacillus sp. in the homogenized extract of Isochrysis sp. with the addition of 5 grams of glucose per litre of physiological solution; the curve 16 is the growth curve of Lactobacillus sp. in the entire extract of Isochrysis sp. sterilized in an autoclave; the curve 17 is the growth curve of Lactobacillus sp. in the entire extract of Isochrysis sp., sterilized in an autoclave with the addition of 5 grams of glucose per litre of physiological solution ; the curve 18 is the growth curve of Lactobacillus sp. in the extract of Isochrysis s ., homogenized and sterilized in an autoclave, and the curve 19 is the growth curve of Lactobacillus sp. in the extract of Isochrysis sp., homogenized and sterilized in an autoclave with the addition of 5 grams of glucose per litre of physiological solution.

The speed of growth of Lactobacillus sp. in the extract of Isochrysis sp. was higher from the first day of inoculation of the bacterium and it wasn't noticeable any advantage with the addition of 5 grams per liter of glucose. The bioavailable sugars were sufficient to ensure the growth of Lactobacillus sp. until at least 4 days after inoculation of this . The present invention has been shown in reference to specific examples thereof, but it must be expressly understood that variations may be made thereto without departing from the scope of the appended claims .

Claims

CLAIMS :

Method of culture of anaerobic bacteria, obligate or facultative, that provides the inoculation of the anaerobic bacteria in an extract, homogenate or entire, of unicellular algae rich in omega - 3 in a solvent under anaerobic conditions. Unicellular microalgae must have a concentration in the solvent, suitable to support the growth of the anaerobic bacteria .

Method of culture of an anaerobic bacterium as claimed in claim 1, further comprising the addition of a complement of sugars to the aforementioned extract of unicellular microalga.

Method of culture as claimed in claim 1 or 2, which also makes unnecessary any subsequent purification step of the bacteria from the growth medium, which in turn has an important nutritional value in lipid content and therapeutic properties due to the presence of omega - 3.

Method of culture of an anaerobic bacterium as claimed in any Claims from 1 to 3 , wherein the mentioned extract is an extract of homogenized unicellular microalgae Nannochloropsis gaditana . Method of culture of an anaerobic bacterium as claimed in claim 4, wherein the mentioned extract of Nannochloropsis gaditana was obtained from a fresh culture of Nannochloropsis gaditana in exponential phase of growth .

Method of culture of an anaerobic bacterium as claimed in claim 1, wherein the mentioned unicellular algae extract is an extract of Isochrysis sp .

Method of culture of an anaerobic bacterium as claimed in claim 6, wherein the mentioned extract of Isochrysis sp . was obtained from Isochrysis sp . lyophilized.

Method of culture of an anaerobic bacterium as claimed in any Claims from 1 to 7 , wherein the mentioned anaerobic bacterium is Lactobacillus sp..

A product containing an entire fermented, comprising in combination both a medium of bacterial culture and a bacterial population grew in it. This is obtained by the method of culture of an anaerobic bacterium, obligate or facultative, as claimed in any Claims from 1 to 7, for which the product is suitable as a source of probiotic and at the same time of the fermented of algae for the growth of these, for nutritional, therapeutic or cosmetic use.

10. Product containing an entire fermented as claimed in claim 9, wherein the mentioned anaerobic bacterium is Lactobacillus sp.. The product is suitable as a source of probiotic and at the same time of the fermented of algae for the growth of the probiotic. This product can be used as a food supplement .

11. Product based on probiotic strains grown in unicellular algae rich in omega - 3 as claimed in any Claims from 1 to 7 which then combine the beneficial properties of the probiotics cultivated and omega - 3 fatty- acids .