CH683103A5 - lactic acid bacteria in symbiotic association, preparation of a concentrate, and their use in the preparation of food products and dietary-therapeutici. - Google Patents

Description

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CH 683 103 A5

Description

The present invention relates to a symbiotic association of live and vital Lactobacillus bulgaricus and Streptococcus thermophilus, in concentrated and dried form, their use in the production of dairy and food products in general for human and animal use, as well as a process for obtain it.

It is known that Lactobacillus bulaaricus and Streptococcus thermophilus can be found in milk and milk products, for example in yoghurt, and that they can serve as a starter culture for these products. The use of a mixture of L. bulgaricus and S. thermophilus to produce cheese and / or yoghurt has long been known. Thus in UK 1 464 617 a process is described for preparing spun paste cheeses of the mozzarella, scamorza and provolone type, in which a mixture of L. bulgaricus and fi is used. thermophilus. Here, however, it is necessary to obtain curd, which is then separated from the whey at a pH of 5.6-6.2 and kept at 33-65 ° C in order to lower the pH until it reaches a suitable value to get the pasta filata. This process is not applicable to soft cheeses, for example gorgonzola, and the starter mixture, even if not expressly stated, is presumably a liquid mixture. FR 2 161 766 describes the preparation of milk derivatives using a liquid mixture of L. bulçiaricus and S. thermophilus. but in this case Lactobacillus casei is also added in concentrated and frozen form.

The addition of L. casei to a mixture of the two lactic bacteria mentioned above is also described in EP 78 813. Here too the mixture of L. bulgaricus and S. thermophilus is in a liquid state, the process is directed towards the preparation of low-fat cheeses and L. casei is added to aid protein hydrolysis.

Finally, DE 2 657 418 describes the preparation of dietary milk derivatives using a mixture of L. bulgaricus and S. thermophilus. but also here the addition of other starters is considered essential, for example L. casei or Lactobacillus acidophilus.

Therefore, in none of these patents is a symbiotic association of L. bulgaricus and S. thermophilus described or mentioned. much less in a concentrated and dried form, nor is there any mention of their possible or possible combination.

However, it is known to use cultures of L. bulgaricus and S. thermophilus in symbiotic association, in liquid or lyophilized form, with cell content of about 500 million / ml or gram. Thus in Nippon Chikusan Gakkai Ho 1982, 53 (3), 161-9 the symbiotic and antagonistic relationships in yoghurt between mixed cultures of L. bulgaricus and S. thermophilus are described. No mention is made of the preparation of these mixtures, their use in the production of dairy products, nor on how these mixtures appear.

In any case, all these cultures in liquid or freeze-dried form must be replicated, before use, on a substrate based on milk or whey, or on other growing medium. A culture is thus obtained, called lactoferment or seroferment, or starter, which is used for the production of dairy or food products, or in addition to feed.

There are also on the market crops of Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association, in concentrated but frozen form (with a number of microorganisms ranging between 10 and 100 billion / gram) which are used directly in the milk to be transformed or in liquid products. All these crops, however, have a limited shelf life dependent on maintenance and at temperatures below 0 ° C they must be kept in liquid nitrogen containers. This results in rather heavy transport and management costs, which then inevitably affect the costs of the finished product.

The aim of the present invention was to provide a suitable combination of live and viable lactic ferments that could be preserved for a long period of time, did not undergo appreciable variations, and could be used in the production of hard or soft cheeses without the need to add other lactic acid bacteria of different species. This object and others have been achieved in accordance with the present invention with live and viable Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association, where the mixture of the two bacteria is presented as a highly concentrated and dried cell mass that can contain up to to 100 billion cells and more per gram, and can be used as such to prepare food products in general and agro-technicians of any type, drugs and diet products without adding bacteria of different species known for this purpose.

The term "foodstuffs in general" as used herein includes all foodstuffs and vegetables indicated for human and animal consumption. As an example, it is only possible to mention, as already mentioned, fermented cheeses and milks, in particular yoghurt, fermented drinks, supplements, and so on. For some of these products it may prove useful or appropriate to add other bacteria or their spores, lactic bacteria, yeasts and molds to the mixture of the invention. This possible addition does not in the least limit or invalidate the originality of the present invention, and this addition will take place in liquid, frozen or lyophilized, concentrated and non-concentrated form.

In this regard, it should be remembered that in the present invention with Lactobacillus bulgaricus and Streptococcus thermophilus we mean the two lactic bacteria per se, as well as all the present and subsequent denominations that will be attributed to these microbial species at a taxonomic level.

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A symbiotic association of live and viable Lactobacillus bulgaricus and Streptococcus thermophilus, in concentrated and dried form, is therefore the object of the present invention, which maintains this association between the two bacteria even in the dried form, remains unaltered for long periods of time at temperatures between 4 ° and 7 ° C, it is simple and easy to transport, and can be used as such to produce hard or soft cheeses, and possibly yoghurt, without having to resort to the addition of lactic acid bacteria of different species as described instead in numerous literature. It is in fact sufficient to vary the ratio between streptococci and lactobacilli in the mixture, to have the desired type of cheese. So to have sweeter cheeses or fermented drinks (yoghurt) the amount of streptococci will be higher than that of lactobacilli, while vice versa to have more acidic cheeses you will have to keep the quantity of lactobacilli higher. The ratio between the two bacteria in the mixture will therefore oscillate within the wider range, and you can have, depending on the type of cheese you want to produce, all combinations of ratios ranging from 1: 100 to 100: 1.

A further object of the present invention is also a method for preparing this symbiotic association of Ibulgaricus and S. thermophilus. which is characterized by the fact that the two bacteria, separately or in association, are inoculated in a culture medium based on milk powder and / or whey powder, malt extract, yeast extract, lactose, potassium phosphate, ammonium sulphate , manganese sulphate, sodium citrate enzymes and urea. It should be remembered that the culture medium is previously sterilized or pasteurized and cooled to the optimum growth temperature of the two microorganisms.

During the incubation, the culture can be possibly kept under stirring, while the pH is kept under control by adding organic or inorganic bases. When fermentation is finished, the culture is cooled and then centrifuged.

The cell concentrate, containing the two forms of live and viable lactic acid bacteria in association, is collected in a cryoprotective substrate made up of substances that guarantee the survival of the cells during drying. This treatment is obtained by freeze-drying or by means of another system which, by eliminating the water from the concentrated mass, guarantees the survival of the microbial cells. The product is used for the preparation of dairy products according to already known technologies.

The following examples serve to further illustrate the invention and must not be considered absolutely limiting.

Example 1

Inoculation preparation

From a culture preserved according to traditional methods (freeze-dried, frozen or liquid form) the inoculum is prepared in whole milk, pasteurized at 90 ° C for 20-30 minutes, incubating at 45 ° C for 3-5 hours.

Intermediate mother preparation

The culture of Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association is inoculated at 1% in a fermenter containing a culture substrate previously pasteurized at 90 ° -100 ° C for 30 minutes and cooled to the temperature of 43 ° -45 ° C, which consists of skimmed milk powder reconstituted 10% in water.

The duration of fermentation is 3-5 hours at the above temperature. At the end of the fermentation period, the culture is cooled with ice water and is ready for the inoculation of the production fermenter.

Fermentation

In this phase it is possible to obtain a liquid product containing the symbiotic association of L. bulgaricus and S. thermophilus in the desired ratio for the two species of lactic ferments. This ratio is then maintained unchanged in the freeze-dried concentrated finished product.

a) 1: 1 ratio

A 3-5% culture substrate is inoculated with the intermediate mother, previously sterilized in a fermenter at 120 ° C for 20 minutes which contains:

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whey powder

60 g / l lactose

10 g / l yeast extract

10 g / l kh2po4

1 g / l

(NH4) 2S04

3 g / l

MnSQ4 • 4H20

0.05 g / l

Before inoculation, the culture medium is brought to pH 6.5 with 25% liquid ammonia and, after sterilization, it is cooled to 45 ° C. Fermentation proceeds for 5 hours at this temperature, possibly stirring the mass, while at one hour intervals the pH is checked to keep it constant at a value of 4.8-6.0. If necessary, 25% ammonia is added. At the end of fermentation the tank is cooled to 4 ° -6 ° C with ice water.

Count no. L. bulgaricus cells: in medium MRS ~ 500 x 106 / ml CFU

Count no. S. thermophilus cells: in medium M17 ~ 500 x 106 / ml CFU

b) Ratio 1: 2.5

Proceed as described under a), with the difference that the cultivation soil includes:

whey powder 60 g / l skimmed milk powder 10 g / l yeast extract 5 g / l

(NH4) 2S04 3 g / l

MgS04 • 7H20 0.05 g / l

MnS04 • 4H20 0.05 g / l

Before inoculation, the culture medium is brought to pH 6.0 with lactic acid. Fermentation temperature: 46 ° C; fermentation duration: 5 hours. At one hour intervals the pH is checked to keep it constant at a value of 4.8-6.0. If necessary, 20% sodium carbonate is added to 20% ammonia.

At the end of fermentation the tank is cooled to 4 ° -6 ° C with ice water.

L. bulgaricus cell count in MRS medium (pH 5.4) ~ 700 x 106 / ml S. thermophilus cell count: in M17 medium ~ 300 x 106 / ml c) Ratio 2.5: 1

Proceed as described under a), with the difference that the cultivation soil includes:

whey powder

60 g / l lactose

10 g / l sodium citrate

5 the yeast extract

5 g / l

(NH4) 2S04

3 g / l kh2po4

1 g / l

MnS04 • 4H20

0.05 g / 1

Before inoculation, the culture medium is brought to pH 6.7 with ammonia. Fermentation temperature: 42 ° C; fermentation duration: 5 hours. At one hour intervals the pH is checked to keep it constant at 4.8-6.5. If necessary, ammonia is added. We then operate as described in a).

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L. bulgaricus cell count: MRS medium (pH 5.4) ~ 300 x 106 / ml S. thermophilus cell count: medium M17 ~ 700 x 106 ml Concentration

The culture broth thus obtained is subjected to a concentration process by centrifugation or ultrafiltration, obtaining a concentrated cell mass having a humidity of about 60% and with a volume equal to about 1/20 of the original.

Homogenization

The concentrate obtained as described above is then homogenized in a cryoprotective solution in the ratio 1: 2 (1 part of concentration ^ parts of solution). This cryoprotective solution may for example consist of 24% lactose, 20% skimmed milk powder, 10% lactose + 5% sodium citrate + 2% monosodium glutamate.

Freeze-drying

The product thus obtained is loaded by means of a volumetric lobe pump into a freeze-drying apparatus in trays, and frozen at a temperature between -30 ° and -40 ° C. The product is then subjected to dehydration up to a residual moisture content of 4-6% at a temperature of 10 ° C.

It should be noted that all operations from fermentation included onwards are carried out under sterile conditions.

Final product

The finished product has an exceptionally high number of lactic ferments (> 1010 - 1011 CFU / g). The cells grown in symbiosis are found in the relationship towards which they were directed in the fermentation phase, and they also maintain the acidification and biochemical characteristics of liquid crops unchanged.

Example 2

Application

The preparation of the yoghurt and the cheeses described below was obtained using doses of lyophilisate concentrate, containing the two forms of live and viable lactic acid bacteria in association, corresponding to about 50 g for the processing of 10 quintals of milk. The finished products obtained meet the market requirements and, in the technological phase, the acidification parameters have been respected which are homologous to those obtained using a normal liquid culture.

Taleggio fi latte is pasteurized for 10 seconds at 72 ° C, then cooled to 37 ° C and then introduced into a cheese boiler. The acidity of the milk is 3.1 SH ° / 50 ml. The symbiotic association of the two bacteria is added at a rate of 2-3 g / q as a lyophilisate concentrate, rennet (titre 1 / 10,000) is introduced in the amount of 40 ml / q and then the milk is left to rest for 20-40 minutes. During this time the milk coagulates. The curd is subjected to breaking with suitable devices and finally extracted from the boiler and placed in special forms for purging. The duration of the processing is about 1-2 hours. To verify the regular progress of the cheesemaking, the acidity of the whey is determined and gradually increases during the stagnation of the curd in the forms, at room temperature. To facilitate the bleeding of the whey, the curd that gradually thickens is turned every half hour at regular intervals. The cheese is brought, after about 5-6 hours of this treatment, into the ripening cells at ~ 5 ° C until maturation has taken place, which, generally, ended after thirty days.

Gorgonzola

The preparation process and technologies are roughly the same as described for Taleggio cheese, with the following exceptions. The coagulation temperature is 30 ° C, the addition of rennet is of the order of 30 ml / q, and the dwell time of the forms in the hot chamber is extended to 10-12 hours (instead of 5-6 hours) . In addition to the symbiotic association of the two bacteria object of the present invention, Pénicillium roqueforti is also added to the milk.

During the maturation of the cheese, after 15 days from the production, the drilling is carried out

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of the paste to allow the molds to develop and to give the blue cheese appearance. The total maturation time is around 60-70 days.

yoghurt

Milk is free from antibiotics and anti-fermentatives (acidity 3.2-3.8 SH ° / 50 ml) and is subjected to pasteurization for treatment at a temperature of 92 ° C for 5 minutes, or at 85 ° in a 5000 I fermenter. C for 30 minutes, or at temperatures between the previous two and at different times. The milk is then cooled to 42 ° -45 ° C and inoculated with the symbiotic association of the invention in the ratio of about 50 g / 10 q and incubated for 2-6 hours. The clot is then broken and cooled and packaged. Alternatively, the milk inoculated with the symbiotic association is packaged and the packs are incubated at 42 ° -45 ° C for 3-6 hours and then cooled in a refrigerator.

DIET-THERAPEUTIC PRODUCT

Live and vital Lactobacillus bulgaricus and Streptococcus thermophilus, in symbiotic association, in concentrated and dried form, prepared in the manner described above, can be used for dietetic and therapeutic purposes both in powder form and in the form of pods or tablets.

The combination of the two lactic ferments can also be used for the enrichment in lactic acid bacteria of dietary foods intended for children and for men, such as, for example, homogenised (based on meat, milk, cheese, fruit, vegetables) or foods for diabetics (jams, chocolates, sweeteners other than sucrose, etc.).

The combination of the two lactic ferments can be mixed with the above mentioned preparations in different proportions. The addition of Lactobacillus bulgaricus and Streptococcus thermophilus in concentrated dried form, in foods, is justified by the fact that this product possesses properties of intestinal flora balancer and stimulator of immunological properties as well as for any anti-tumor properties and stimulation of natural defenses body.

Preparation example:

- Homogenized for infancy: mix 1 gram of live and viable Lactobacillus bulgaricus and Streptococcus thermophilus, in symbiotic association, in concentrated and dried form, with 50-100 g of homogenate.

- Tablets: mix 1 gram of the two lactic ferments in object alive and vital, in symbiotic association, in a concentrated and dried form, with the excipients necessary for the formulation of the tablet.

- Cereals: mix 1 gram of the two lactic ferments in object alive and viable, in symbiotic association, in a concentrated and dried form, with 50-100 g of cereals (e.g. bran).

Example 3

a) Preparation of a piglet feed

- Supplement

The following ingredients are introduced and mixed in a double-cased (or V-shaped) mixer of adequate capacity:

1) support consisting of lactose or whey powder or other

2) Lactobacillus bulgaricus and Streptococcus thermophilus live and viable in symbiotic association, in a dried concentrated form, in quantities such as to obtain, for example, a microbial load of 2 billion per gram of supplement.

- Feed

The integrator thus obtained is introduced, in the desired quantity, into a horizontal mixer and is mixed with the other constituents of the feed in the proportions listed below:

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- skimmed milk powder

10%

- whey powder

5%

- soy flour

10%

- corn flour

20%

- fish meal

5%

- barley flour

5%

- oatmeal

2.5%

- barley flakes

20%

- oatmeal

5%

- bran

15%

- mineral salts

1%

- vitamin mix

1%

- supplement with lactic ferments

0.5%

b) Preparation of an integrated compound feed replacement for milk for calves which also obviates the use of antibiotics

- whey powder 85% coconut oil 8%

- fat 4% cereal flour 2% mineral vitamin supplement 0.5%

- lactic acid bacteria 0.5% pH 5.0 after reconstitution to 10% in water.

Claims (13)

claims 1. Process for preparing a freeze-dried live and viable Lactobacillus bulgaricus and Streptococcus thermophilus concentrate in symbiotic association, characterized in that the two microbial species are inoculated together or separately in a culture medium based on powdered milk and / or whey powder , malt extract, yeast extract, lactose, potassium phosphate, ammonium sulphate, manganese sulfate, sodium citrate, enzymes and urea, and incubated under controlled temperature and pH conditions, and / or under stirring; the grown culture is cooled and centrifuged, and the cell concentrate containing the two forms of live and viable bacteria in association is collected in a cryoprotective substrate, and finally it is homogenized and dried. 2. Process according to claim 1, characterized in that before the inoculation the medium is brought to a pH between 6 and 7 with basic or acidic, inorganic and / or organic substances, sterilized for about 20 minutes and then cooled to 42-46 ° C. 3. Process according to claim 1 or 2, characterized in that the culture medium contains whey powder, lactose, extract, yeast, ammonium sulphate, potassium phosphate, manganese sulfate, powdered milk, sodium citrate. Method according to one of claims 1 to 3, characterized in that during the incubation the pH value is checked every hour to maintain it between 4.5 and 6.5 by means of ammonia or other organic and inorganic basic substances . Method according to one of claims 1 to 4, characterized in that the incubation lasts from 3 to 6 hours and the cooling is carried out at 4-6 ° C by means of ice water. Method according to one of claims 1 to 5, characterized in that the cell concentrate and the cryoprotective substrate are mixed in the ratio oscillating between 1: 1 and 1: 2. 7. Process according to claim 5, characterized in that the cryoprotective substrate consists of substances which guarantee the survival of the cells during drying, in particular 24% lactose, 20% skimmed milk powder, 10% lactose + citrate sodium 5% + monosodium glutamate 2%. 8. Live and vital lactic ferments in symbiotic association, in concentrated and freeze-dried form, obtained 7 5 10 15 20 25 30 35 40 45 50 55 CH 683 103 A5 according to the process of one of claims 1 to 7, for use in the production of food products for human and animal use. Live and viable Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association, in concentrated and freeze-dried form, obtained according to one of claims 1 to 7, for use in the production of all food, agro-technical and vegetable products for human consumption and animal and for the production of dietetic products and drugs. Live and viable Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association, in concentrated and freeze-dried form according to claim 9, together with other bacteria or their spores, lactic bacteria, yeasts and molds, in liquid, frozen or freeze-dried, concentrated and non-concentrated form , for use in the production of food, agro-technical and vegetable products. Live and vital Lactobacillus bulgaricus and Streptococcus thermophilus in symbiotic association, in a concentrated and freeze-dried form according to claim 9, characterized in that the ratio in the mixture between the two ferments oscillates between 1: 100 and 100: 1. 12. Use of the symbiotic association of Lactobacillus bulgaricus and Streprococcus thermophilus in concentrated and freeze-dried forms prepared according to the procedure of one of claims 1 to 7 to produce food, agro-technical, vegetable, dietetic products and drugs for human and animal use. Use according to claim 12, characterized in that the food products are selected from the group comprising hard and soft cheeses, fermented milks, fermented drinks, yoghurt and supplements. 8