FR2609044A1 - LACTIC FERMENTS IN SYMBIOTIC ASSOCIATION AND THEIR USE IN THE PREPARATION OF DAIRY, CHEESE AND FOOD PRODUCTS IN GENERAL - Google Patents

Abstract

LACTOBACILLUS BULGARICUS AND STREPTOCOCCUS THERMOPHILUS LIVE AND VITAL IN SYMBIOTIC ASSOCIATION, IN CONCENTRATED AND DRIED FORM, ARE ADVANTAGEALLY USED IN THE PRODUCTION OF DAIRY-CHEESE PRODUCTS FOR HUMAN AND ANIMAL USE. A METHOD FOR THE PREPARATION OF THIS SYMBIOTIC ASSOCIATION OF TWO MILK BACTERIA AND A PROCESS FOR THE PREPARATION OF DAIRY-CHEESE AND FOOD PRODUCTS IN GENERAL, BY MEANS OF THIS ASSOCIATION.

Description

The present invention relates to a symmetrical association

  biotic of Lactobacillus bulgaricus and Streptococcus

  active and vital thermophilus, in concentrated and dry form

  their use in the production of dairy products-

  cheese and food in general for human and animal use

  wrong, as well as a method for obtaining this association.

  We know we can find Lactobacillus bulqari-

  cus and Streptococcus thermophilus in milk and in milk derivatives, for example in yogurt, and which they can serve as a starter culture (called

  following "starter") for these products.

  The use of a mixture of L. bulqaricus and S. thermo-

  philus for the production of cheese and / or yogurt has been known for a long time. Thus, UK 1,464,617 describes a process for the preparation of cheeses of the spun type, such as mozzarella, scamorza and provolone (it should be noted that the cheeses mentioned in this text are always typical Italian cheeses), in which

  employs a mixture of L. bulgaricus and S. thermophilus.

  Here however it is necessary to obtain curd which is then

  separated from the serum at a pH of 5.6 to 6.2 and maintained at 33 -

   C so as to lower the pH until when it reaches a value suitable for obtaining a spun dough. This process is not applicable to soft cheeses, for example gorgonzola and the starter mixture is probably a liquid mixture, although this

  has not been expressly declared.

  Document FR 2.161.766 describes the preparation of

  riveted milk using a liquid mixture of L. bul-

  garicus and S. thermophilus, but in this case we add é-

  also Lactobacillus casei in concentrated form and

  frozen. The addition of L. casei to the mixture of the two bac-

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  above mentioned milk series is also described

  te EP 78813. Here also the mixture of L. bulgaricus and S. thermophilus is in a liquid state, the process is intended for the preparation of lean cheeses and L. casei is added in order to help the hydrolysis of proteins.

  In DE 2,657,418 we finally describe the preparation of

  dietary rivets of milk using a mixture of L. bulgaricus and S. thermophilus, but also in this case we consider essential the addition of other starters, for

  example L. casei or Lactobacillus acidophilus.

  None of these patents, therefore, describes or mentions

  is a symbiotic association of L. bulgaricus and S. thermophilus, much less in concentrated and dried form, and there is also no mention of a

  possible or possible combination of these bacteria.

  However, we already know the possibility of using

  cultures of L. bulqaricus and S. thermophilus in combination

  Symbiotic ciation, in liquid or lyophilized form, with a cell content of around 500 million / ml or gram. Thus the document Nippon Chikusan Gakkai Ho 1982,

  53 (3), 161-9 describes the symbiotic and antagonistic relationships

  nists in yogurt between mixed cultures of L.

bulgaricus and S. thermophilus.

  No mention is made of the preparation of these mixtures, their use in the production of

  dairy-cheese products and their preparation.

  Anyway, all these cultures in liquid form

  of or freeze-dried should be repeated before use

  age, on a substrate based on milk or milk serum, or on another cropland. We thus obtain a culture, called lactoferment or seroferment, or starter,

  which is used for the production of dairy-cheese products

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  gers or is added to the feeds and feed.

  There are also crops in the trade

  of Lactobacillus bulgaricus and Streptococcus thermo-

  philus in symbiotic association, in concentrated form

  very but frozen (where the number of microorganisms varies between 10 and 100 billion per gram) which are used directly in milk which must be processed or in liquid products. However, all of these cultures have a limited shelf life depending on the maintenance and at temperatures below

  0 C they must be kept in nitrogen containers

  liquid. This translates into quite heavy transport and management costs, which inevitably have an in

  negative impact on the prices of the finished product.

  The object of the present invention was to make available an appropriate combination of vital and vital lactic ferments which was able to keep

  for a long period of time, does not undergo varia-

  remarkable and could be used in production

  hard or soft cheeses without the need to have there-

  add other milk bacteria of different species.

  This object and others have been achieved according to the present invention by the Lactobacillus bulgaricus and Streptococcus thermophilus alive and vital in symbiotic association, where the mixture of the two bacteria appears as a very concentrated and shaped mass of cells.

  dried which can contain up to 100 billion cells

  and even more per gram and which can be used as is for the preparation of cold dairy products

  magers, food in general and intended for the agricultural-

  ture and zootechnics of any type without adding bacteria of different species known for this purpose.

  The term "dairy and cheese and food products"

  res in general "as used herein, includes all food, dietetic, pharmaceutical, agricultural and zootechnical products and plant products intended for human and animal consumption. can mention, like

  already specified, fermented cheeses and milks, in particular

  include yogurt, dietetic and pharmaceutical products, mangeages and feed, products

  integration and so on. Regarding what-

  some of these products, it might prove useful or suitable to add to the mixture of the invention other

  bacteria or their spores, milk bacteria,

  vain and mold. This possible addition does not

  does not mite or in any way invalidate the originality of the present invention and this addition will take place in liquid form,

  frozen or lyophilized, concentrated and not.

  In this regard, it should be remembered that in the present

  vention by Lactobacillus bulgaricus and Streptococcus

  thermophilus means the two bacteria of milk consi-

  separately, as well as all the pre-

  which will be attributed to these species

  these microbials at the taxonomic level.

  The object of the present invention is a symbiotic association of Lactobacillus bulgaricus and Streptococcus thermophilus alive and vital, in concentrated and dried form, which maintains this association between the two bacteria also in its dried form, remains inal

  tered for long periods of time at temperatures

  erasures varying between 4 and 7 C, allows simple transport

  full and easy and can be used as is for the production of hard or soft cheeses, and possibly

  yogurt, without having to resort to the addition of bacteria

  milk of different species, as described in a large part of the existing literature. It is indeed sufficient to vary the ratio between the streptococci and the lactobacilli in the mixture, to have the desired type of cheese. This is how to have milder cheeses or fermented drinks (yogurt) it will be necessary to aul ment the amount of streptococci compared to that of

  lactobacilli, while on the contrary, to have fro-

  more acidic mages will need to use a larger amount

  lifting of lactobacilli. The ratio of the two bacteria in the mixture will vary in the widest range and

  we can have, depending on the type of cheese we want

  re produce, all combinations of reports ranging from

1: 100 to 100: 1.

  Another object of the present invention is also

  a method for the preparation of this sym-

  biotic of L. bulgaricus and S. thermophilus, which is characterized in that the two bacteria, separately or in combination, are inoculated in a cultural field based on powdered milk and / or powdered serum, extract

  malt, leaven autolysate and other cultured substrates

  suitable salts, to which have been added the appropriate mineral salts for their development, and possibly

  enzymes and urea. It should not be forgotten that the ter-

  rain cultural is previously sterilized or pasteurized and cooled to the optimum temperature for growing

two microorganisms.

  During the incubation the culture can be possible-

  stirring, while controlling the

  pH by the addition of organic or inorganic bases.

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  When the fermentation is finished, the culture is re-

cooled and then centrifuged.

  The cell concentrate, containing the two forms of living and vital lactic acid bacteria in combination, is collected on a cryoprotective substrate formed of substances which guarantee the survival of the cells during drying. This treatment is obtained by freeze-drying or by another method which, by eliminating the mass

  concentrated, water, ensures the survival of mid-cells

  crobians. The product is used for the preparation of dairy-cheese products according to already known technologies. The following examples serve to further illustrate the invention and should not be considered in any way

limiting.

Example 1

  Preparation of the inoculation substance

  From a culture preserved according to traditional methods-

  nelles (lyophilized product, frozen or in liquid form), the inoculation is prepared in whole milk, pasteurized at C for 20-30 minutes, by incubation at 45 C

for 3-5 hours.

  Preparing the intermediate mother

  Cultivation of Lactobacillus bulgaricus and Streptococ-

  cus thermophilus in symbiotic association is inoculated at a rate of 1% in a fermenter containing a cultural 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 at 10% in water. The duration of the fermentation is 3-5 hours at the temperature specified above. At the end of the

  fermentation, the culture is cooled with cold water

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  ready and inoculated with the production fermenter

  tion. Fermentation During this stage we have the possibility of obtaining a liquid product containing the symbiotic association

  of L. bulgaricus and S. thermophilus according to the desired report

  d for the two species of lactic ferments. This report

  is then kept unaltered in the concentrated finished product

very lyophilized.

  a) 1: 1 ratio With the intermediate mother, inoculate at 3-5% a cultural substrate previously sterilized in a fermenter at 120 C for 20 minutes, which contains: powdered serum 60 g / l lactose 10 g / l extract sourdough 10 g / l KH2PO4 g / l (NH4) 2SO4 3 g / l MnSO4. 4H2O 0.05 g / l Before inoculation, the cropland is brought to a pH of 6.5 with 25% liquid ammonia and, after

  sterilization, it is cooled to 45 C.

  Fermentation continues for 5 hours at this

  temperature, the mass is possibly agitated and at in-

  one hour intervals the pH is checked with a view to

  keep constant at a value of 4.8-6.0. If necessary

  re, 25% ammonia is added thereto. At the end of the fermentation, the tank is cooled to 4-6 C with ice water. Counting n L. bulgaricus cells: in MRS field (pH 5.4) É 500x106 / ml CFU Counting n S. thermophilus cells: in M17 field - 8 - 500x10 / ml CFU b) Ratio 1: 2.5 We will proceed with the manner described in point a), with the difference that the cropland contains: powdered serum 60 g / 1 skimmed milk powder 10 g / 1 leaven extract 5 g / 1 (NH4) 2SO4 3 g / l MgSO 4 7H2 O0.05 g / l MnSO4. 4H2O 0.05 g / i Before inoculation, the cropland is brought to a pH of 6.0 with lactic acid. Temperature

  fermentation: 46 C; duration of fermentation: 5 hours.

  At one hour intervals the pH is checked with a view to

  keep constant at a value of 4.8-6.0. If necessary

  re, add 20% sodium carbonate in ammonia

at 20%.

  At the end of fermentation the tank is cooled

at 4 -6 C with ice water.

  Counting n L. bulgaricus cells in MRS ground (pH 5.4), 700x10: ml Counting n S. thermophilus cells: in M17 ground 300x10 / ml We will proceed as described in point a), with the difference that the ground cultural contains: powdered serum 60 g / 1 lactose 10 g / 1 sodium citrate 5 g / 1 leaven extract 5 g / 1 (NH 4) 2S4 3 g / 1 KH2PO4 1 g / i MnSO4. 4H2O 0.05 g / 1 -9- Before inoculation, the cropland is brought

  at pH 6.7 with ammonia. Farm temperature

  tation: 42 C; duration of fermentation: 5 hours. To inter-

  one hour intervals pH is checked for maintenance

  nir constant on the value of 4.8-6.5. If necessary, add ammonia. We operate as described

in point a).

  Counting n L. bulgaricus cells: MRS field (pH 5.4) - 300x10 / ml Counting n S. thermophilus cells: field M17 700x10 / ml Concentration The culture broth thus obtained is subjected to a

  concentration process by centrifugation or ultrafil-

  trage so as to obtain a concentrated cell mass

  with a humidity of around 60% and a volume equal to

about 1/20 of the original.

  Homogenization The concentrate obtained in the manner described above is then homogenized in a cryoprotective solution according to the ratio 1: 2 (1 part of concentrate: 2 parts of solutions). This crioprotective solution can for example be formed from 24% lactose, skimmed milk powder

  at 20%, lactose 10% + sodium citrate 5% + glutamate mono-

sodium 2%.

Lyophilization

  The product thus obtained is charged by a vo-

  lumimetric with lobes in a lyophilization apparatus, in cuvettes, and frozen at a temperature varying between minus

  30 C and less 40 C. The product is then subjected to dehy-

  dratation up to a residual moisture content of

4-6% at a temperature of 10 C.

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  It should be noted that all operations starting from the fermentation included are carried out under conditions

of sterility.

  Finished product The finished product has an exceptionally high number of lactic ferments (> 10 -101 CFU / g). The cells which have grown in symbiosis are found in the report to which they were addressed in the step

  of fermentation and, moreover, they maintain inal-

  the acidification and biochemical characteristics

ques specific to liquid cultures.

Example 2

Application

  The preparation of yogurt and cheeses described below

  after was obtained using doses of freeze-dried

  concentrated, containing the two forms of lactic acid bacteria

  ques lively and vital in combination, corresponding to approximately 50 g for the treatment of 10 q of milk. Products

  finishes obtained meet the conditions required on the market

  ché and, during the technological stage, we respected the

  acidification parameters which are homologous to those obtained

  kept using a normal liquid culture.

  Taleggio (Italian soft cheese) The milk is pasteurized for 10 seconds at 72 C; then cooled to 37 C and introduced into a cheese boiler. The acidity of the milk is 3.1 SH / 50

  ml. We add the symbiotic association of the two bacteria

  ries due to 2-3 g / q in lyophilized concentrated form

  trée, the milk curd (title l / 10,000) is introduced in the amount of 40 ml / q and the milk is left to stand for

  -40 minutes. During this period the milk coagulates.

  The curd is subject to breaking by means of devices

- he -

  it is finally extracted from the boiler and put into appropriate molds where it is subjected to the purge operation. The working time is about 1-2 hours. In order to check the regular development of the caseification, the acidity of the serum, which increases during the curdling stasis in the molds, is checked at room temperature. To promote the flow of serum, the curd which gradually curdles is turned every half hour at regular intervals. The cheese, 5 to 6 hours after this treatment, is brought to the ripening chambers at 50C until the end of the ripening, which normally takes place after

thirty days.

  Gorgonzola (Italian cheese close to Roquefort)

  The preparation process and the technologies are

  roughly those described for Taleggio cheese, ex-

  except the following. The coagulation temperature is 30 ° C., the addition of the milk curd is of the order of 30

  ml / q and the pause time of the molds in the hot chamber

  from is 10-12 hours (instead of 5-6). In addition to the as-

  symbiotic association of the two bacteria which are the subject of the present invention, milk is also added with

Penicillium roqueforti.

  While the cheese is roasting, after 15 days of

  production, holes are made in the dough to

  put mold to grow, which gives the cheese the marbled appearance. Total maturation time

is around 60-70 days.

  Yogurt We carry milk free of antibiotics and products

  anti-fermentation (acidity 3.2-3.8 SH / 50 ml) in an iron

  liar of 5000 1 and subjected to the treatment of pasteur-

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  temperature at 92 C for 5 minutes,

  or at 85 C for 30 minutes, or at temperatures

  tures varying between the two previous temperatures, for different periods of time. The milk is then cooled to 42 ° -45 ° C. and inoculated with the symbiotic association of the invention at a rate of approximately g / 10 q and incubated for 2 to 6 hours. The coagulum is broken and cooled and conditioned. Alternatively,

  the milk inoculated with the symbiotic association is con-

  added and the conditioned product is incubated at 42 ° -45 ° C. for 3-6 hours and then cooled in a refrigerating chamber.

DIETETIC-THERAPEUTIC PRODUCT

  Lactobacillus bulgaricus and Streptococcus thermophilus alive and vital, in symbiotic association, in the form

  concentrated and dried, prepared as described above

  above can be used for dietary and therapeutic purposes either in powder form or in the form

cachets or tablets.

  The combination of the two lactic ferments can also be used for the enrichment of lactic bacteria with dietetic foods intended for children and adults such as, for example, homogenized products (based on meat, milk, cheese, fruits, vegetables, etc.) or foods for diabetics

  (jams, chocolates, sweeteners other than saccha-

pink, etc.).

  The combination of the two lactic ferments can be mixed with the preparations indicated above in different proportions. The addition of Lactobacillus

  bulgaricus and Streptococcus thermophilus in con- form

  centered dried, in food, is justified by the

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  fact that this product has balancing properties

  intestinal flora and stimulant of pro-

  immunological properties and also possible proprieties

  tees against tumors and stimulation of natural defenses

  body turalles. Example of preparation: - Homogenized intended for children: mix 1 g of Lactobacillus bulgaricus and Streptococcus thermophilus alive and vital, in symbiotic association, in the form

  concentrated and dried, with 50-100 g of homogenized.

  - Tablets: mix 1 g of the two vital and vital lactic ferments in question, in symbiotic association, under

  concentrated and dried form, with the necessary excipients

  res to the preparation of the tablet.

  - Cereals: mix 1 g of the two vital and vital lactic ferments in question, in symbiotic association, in concentrated and dried form, with 50-100 g of cereals

(e.g. sound).

Example 3

  a) Preparation of feed intended for small pigs - Integration product In a double jacket mixer (or V-shaped) of suitable capacity, the following ingredients are introduced and mixed: 1) support formed from lactose or milk serum in powder or other 2) Lactobacillus bulgaricus and Streptococcus thermophilus alive and vital in symbiotic association, in dried concentrated form, in sufficient quantity to obtain, for example, a microbial load of 2 billion

  per gram of integration product.

- Provenses

- 14 -

  The integration product thus obtained is introduced, according to the desired quantity, into a horizontal mixer

  and it is mixed with the other products making up the

  sold in the proportions indicated below: - skimmed milk powder 10% - milk serum powder 5% - soy flour 10% - corn flour 20% - fish meal 5% - barley flour 5% - oatmeal 2.5% - barley flakes 20% - oat flakes 5% - bran 15% - mineral salts 2% - vitamin mixture 1% - integration product with lactic ferments 0.5% b) Preparation of an integrated compound feed replacement for milk, intended for calves, which also eliminates the use of antibiotics 85% milk powder serum - 8% coconut oil - 4% fat - 2% cereal flour - product vitamin mineral integration 0.5% - lactic acid bacteria 0.5%

  pH 0.5 after reconstitution at 10% in water.

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Claims (16)

R E V E N D I C A T I ON S   1. Vivid and vital lactic ferments in combination   symbiotic in concentrated and dried form, for use   serve in the production of dairy-cheese and food products in general for human and animal use.   2. Lactic acid bacteria according to claim 1, class-   "Lactobacillus bulgaricus and Streptococcus ther-   mophilus "(from" Manual Bergey's, 1974), including all   are the present and subsequent denominations which are   will eventually be attributed to these microbial species- born at the taxonomic level.   3. Lactobacillus bulgaricus and Streptococcus thermophi-   read alive and vital in symbiotic association, in concentrated and dried form, to be used in the production of all food products, intended for agriculture   and zootechnics and plants, used for food-   human and animal, such as hard or soft cheeses, fermented milks, fermented drinks, yogurt, dietetic and pharmaceutical products, feed, products integration and so on.   4. Lactobacillus bulgaricus and Streptococcus thermophi-   read alive and vital in a symbiotic association, in form   concentrated and dried, possibly together with other bacteria or their spores, milk bacteria, sourdoughs and molds, in liquid, frozen or   freeze-dried, concentrated and not, to be used in production   tion of food products, agricultural products   culture and zootechnics and plants as claimed cation 3.   5. Lactobacillus bulgaricus and Streptococcus thermophi-   read alive and vital in a symbiotic association, in form   concentrated and dried, characterized in that the - 16 -   port between the two ferments in the mixture varies   in the widest interval, so that we can   ra have, depending on the type of dairy-cheese, food, intended for agriculture and animal husbandry, plants, etc. that we want to prepare, all the   shift ratios ranging from 1: 100 to 100: 1.   6. Process for the preparation of a lyophilized concentrate of Lactobacillus bulgaricus and Streptococcus thermophilus   in symbiotic association according to claims 1 to   5, characterized in that the two microbial species   are inoculated together or separately in a growing ground   tural based on powdered milk and / or powdered serum, malt extract, sourdough extract and other suitable cultural substrates, optionally comprising mineral salts necessary for their development, enzymes and urea, and incubated under conditions controlled temperature and pH, and, if necessary, with stirring; the raw culture is cooled and centrifuged, and the cell concentrate containing the two forms of live and vital bacteria in association is collected in a cryoprotective substrate; finally homogenization and drying are carried out. 7. Process for the preparation of a lyophilized concentrate of Lactobacillus bulgaricus and Streptococcus thermophilus   in symbiotic association according to claims 1   to 5, characterized in that the two microbial species   are respectively inoculated in two separate reservoirs   res and incubated according to the method of claim 6,   individually concentrated and lyophilized, and mixed   managed after according to the desired report.   8. Process for the preparation of a lyophilized concentrate of Lactobacillus bulgaricus and Streptococcus thermophilus - 17 - 2609044 - 17 -   in symbiotic association according to claims 1 to   , characterized in that the two microbial species   are respectively inoculated in two separate reservoirs   res and incubated according to the method of claim 6, mixed in liquid form or concentrated according to the ratio   desired port and finally lyophilized jointly.   9. Method according to claim 7, characterized in that the soil is brought, before inoculation, to a pH varying between 6 and 7 by means of basic or acidic, inorganic and / or organic substances, sterilized for about 20 minutes at 110-120 C and then cooled to 42o-46oC.   10. Method according to claims 6 and 9, character-   se in that the cropland contains: serum in   dre, lactose, leaven extract, ammonium sulfate,   KH2PO4, MnSO4. 4:20 am, "corn steep", skimmed milk for   dre, MgSO4 7:20, sodium citrate, or other substrates   organic and inorganic suitable for this purpose.   11. Method according to claims 6 to 10, character-   in that during the incubation we control all   hours the pH value in order to keep it on a   between 4.5 and 6.5 by adding any-   ammonia or other basic substances or ganic and inorganic.   12. Method according to claims 6 to 11, 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.   13. Method according to claims 6 to 12, characterized   in that the cell concentrate and the cryoprotective substrate are mixed according to a ratio varying between 1: 1 and 1: 2. - 18 -   14. Method according to claim 13, characterized in that the cryoprotective substrate is formed of substances guaranteeing cell survival during drying, namely: 24% lactose, 20% skimmed milk powder, 10% lactose + sodium citrate 5% + monosodium glutamate that 2%.   15. Method according to claims 6 to 14, characterized   in that the final drying is carried out by lyophilization or by any other method which, by eliminating   the water of the concentrated mass, ensures the survival of these microbial cells.   16. Use of the symbiotic association of claims   1 to 5, prepared according to the process of claims   6 to 15, for the production of dairy-cheese and food products in general for human and animal use according to claim 3.