DE2322146C2 - Process for the manufacture of a mass which can be used as inoculum in the manufacture of starters and fermented milk products - Google Patents

Description

2. The method according to claim 1, characterized in that one uses a component (d) which Contains whey that has been heated to between 85 and 100 ° C for 10 to 30 minutes.

3. The method according to claim 2, characterized in that one uses a component (d), the one Contains whey which is heated to a temperature between 85 and 100 ° C for 10 to 30 minutes and in a Centrifuge has been cleared.

4. The method according to any one of claims 1 to 3, characterized in that the component (0

! 1 uses skimmed milk.

5. The method according to claim 4, characterized in that one uses 5 to 10 vol .-% skimmed milk.
6. The method according to any one of claims 1 to 5, characterized in that a mixture containing equimolar amounts of sodium hydrogen phosphate and potassium hydrogen phosphate is used as the buffer mixture.

The present invention relates to a method for producing a mass used as inoculum in the Making starters and fermented dairy products is useful. This mass contains at least a strain of lactose fermenting bacteria and is obtained by having

a) a culture of the bacterial strain (strains) used in a culture medium containing protein breeds and, if desired,

b) subsequently in the culture medium, in which the protein that is still present may be dissolved in a Form has been transferred, the said mass constricts and

c) subjecting the concentrate to such a treatment that it is brought into a stable condition.

In cheese making, milk casein is made insoluble, and subsequently it is made insoluble along with the Milk fat separated from the main part of the aqueous phase (whey). This insolubilization is achieved by the action of a coagulation enzyme and / or by lowering the pH of the aqueous phase.

so The lowering of the pH value can be done by adding an acid or acid-forming substances, but is mostly caused by the action of a starter.

A starter is a culture of one (pure culture) or several (mixed culture) strains of bacteria that Are able to convert milk sugar into lactic acid. In the Netherlands, common catfish becomes one mixed culture used.

Such a starter is made in a small amount, on the order of 1% of the milk volume of cheese milk added, and the starter bacteria begin to multiply in it. This process favors the desired conditions for the curdling of the milk and / or for the subsequent exudation of the whey from the quark during its treatment - both processes take place under the influence of the lowering of the pH value as a result of lactic acid formation - and the desired degree of ripeness of the cheese as a result the effect of the enzymes formed.

According to one of the usual methods, a starter is produced by inoculating a suitable medium with a produced small amount of the desired bacteria and incubating.

In general, the medium used is highly pasteurized skimmed milk and the temperature of the incubation is about 20 ° C. As already stated, the pH value of the medium is determined by the formation of lactic acid degraded. In addition, other changes occur in the medium (others are formed Reaction products). All of this together causes a decrease in the growth rate of the bacteria. The starter is preferably used immediately after its manufacture; otherwise it has to be cooled down and stored at a low temperature, otherwise the activity will be reduced, which means that the

Decreases the rate at which bacteria build up acid during the cheese making process will.

It is known that the bacterial cultures which are required for cheese production are specialized in Centers to breed. These starters are then in frozen form either after constricting or as well Distributed without constriction and even in a dried form. Drying can be achieved by evaporation of the Water in a stream of hot air or by subliming ice at a very low temperature under reduced Printing. In general, the lactic acid formed is removed before the bacterial culture is dried neutralized; if desired, however, this can already be done during incubation. This neutralization measure is very important, otherwise the lactic acid content can become too high in the narrowing step. Moreover, after specific additives have been added, the initiator can be subjected to certain treatments wejien, initially in a centrifuge, for example a Baktofuge, in which it is divided into a portion, which contains practically all bacterial cells and is divided into a proportion that is relatively line-free. Of these portions, only the first one needs to be frozen. Such frozen cell concentrates are without one or more subsequent vaccinations ready for use in the manufacture of the starter or for the direct addition to cheese milk instead of the starter. Since other possible forms in which the starter can be exercised supplies must be vaccinated at least once to get a starter with satisfactory effectiveness As a result, the use of frozen cell concentrates becomes an important one Saving in manufacturing costs achieved. On the other hand, however, such cultures do not contribute significantly the yields in cheese production, while starters that have not been separated in a centrifuge, make an effective contribution. For this reason, the frozen starter concentrates must not be too expensive be.

It is obvious that skimmed milk can be used as a culture medium in the centralized production of starters as it is the case in cheese selenium, can be used. This is disadvantageous in that in the concentrate or milk protein is also accumulated in the dried starter powder, so that ultimately the number of Bacteria per gram not big enough 1st. The required product volume is increased, so that problems of everyone Kind of like difficulties in storage and transportation, causing an increase in cost. This can be avoided by neutralizing the milk protein prior to the concentration step is dissolved with sodium hydroxide and by adding, for example, sodium chloride. This method is expensive, since in this case the protein is not transferred into the cheese, but it is advantageous in this respect. than on this catfish the concentration can be done by means of a centrifuge. With another, but nevertheless expensive method, N.J. Berrldge and P. Wilson [18 Int. Dairy Congr. (1970) IE 276] a corresponding improvement through prior hydrolysis of the protein with pepsin. Also the Hydrolysis by trypsin and papaln is mentioned.

Moreover, it is possible. To use culture media ("synthetic media") which, in addition to a carbon source - for example glucose - and salts also yeast extract, spring spring water or similar substances contain as growth-promoting substances. Such stalks can also contain whey and even skimmed milk can be added. However, such mixtures have the disadvantage that they cause changes in the microwave The composition of the culture can cause the growth of certain bacteria (variants) promote at the expense of others. In mono-cultures these can be slowly growing variants. In Mixed cultures can have changes in proportion to the number of different ones originally present Species and tribes enter. In addition, and despite the presence of stimulants, there is growth In such media often low, while the stool are relatively expensive. In this matter the use of media consisting predominantly of whey was also recommended. Without subsequent delivery however, only an insufficient amount of nitrogen can be extracted from the media mentioned by additional nitrogen sources be assimilated.

J. M. Lattey [N.Z.J. Dairy ScI 3, (1968), 35] found that the slow development of starters in whey by the addition of yeast extract has been improved. In Dutch patent application 70 05 485, the equivalent U.S. Patent Application No. 818417 filed April 22, 1969 (Miles Laboratories Inc.) discloses the use of a Medium recommended, which is made by dissolving whey and milk powder in water, which Treat the solution with protease and add spring water.

E. G. Pont and G. L. Holloway [Austral. J. Dairy Technol. 23, (1968), 22] recommended the use of Whey to which yeast extract and tryptone had been added. In such a medium, sodium lactate acts as a factor limiting bacterial growth.

Such media also contain substances that stimulate the growth of certain bacteria In mixed cultures to changes in the desired proportions and thus to deviations in the Starters can lead. However, this problem is not encountered in the monocultures referred to above refer to publications mentioned.

In order to obtain a large number of bacteria per liter, the one formed during cultivation is often used Lactic acid neutralized (cf. Dutch patent application 70 05 485, that of US patent application No. 8 18 417 of April 22, 1969 equivalent 1st). The disadvantages of this system are, among other things, an increased probability, that homologous bacteriophages develop, and possibly changes in the ratio between the different bacteria. In addition, undesired salts can form during neutralization form.

It has been found that this disadvantage is eliminated according to the invention with the measures of claim 1 can be. Claims 2 to 6 name embodiments of the invention.

This procedure makes it possible to obtain a concentrated culture for lactic acid bacteria which, like g

In the case of the usual mass starters, they can be introduced directly into the cheese mixture without prior cultivation can. In addition, it is a particular advantage that the ratio of the number of different tribes and

3 I.

Variants of bacteria present in the culture according to the invention are the same as in the normal bulk starters, while the activity is sufficiently high for cheese production. In addition, and in contrast to cultures obtained by known methods, the cost of the culture medium according to the invention is low and the number of bacteria per unit weight is ultimately high.
The aqueous solution used according to the invention under (d) of claim 1 is therefore whey, dilute whey or a whey with a low lactose content or an aqueous dispersion of whey powder. If the carbohydrate content is too low, it is supplemented with lactose, glucose or another assimilable carbohydrate (see S. Orla-Jensen, The Lactic acid bacteria, Copenhagen, 1919; and idem, supplementary volume, Copenhagen, 1943).

Preferred catfish is used as a solution based on whey, a whey 10 to 30 minutes has been heated to a temperature of 85 ° C to 100 ⁰ C long. The whey can be effectively clarified by centrifugation after heating.

In order to prevent changes in the usual composition that ultimately results in a solution Enter whey base, an aqueous dispersion is used according to (e) of claim 1. There is only one small amount of this dispersion is required. Preferably 5 to 10 vol. 96 skimmed milk or an equivalent Amount of an aqueous dispersion of skimmed milk powder used, although also casein or a Caselnat or soy protein are suitable for the intended purpose.

The buffer mixture used according to the invention prevents premature inhibition of bacterial growth.

- ° Various known substances can be used as a buffer mixture as long as they are not harmful Effect on bacteria and as long as they are from a toxicological point of view in food are acceptable.

The buffer mixture is preferably used in the form of an aqueous solution. The alkali salts of orthophosphoric acid, especially an aqueous solution of equimolar amounts of disodium hydrogen phosphate and Kalumdlhydrogenphosphat are very suitable agents.

The addition of phosphates to dairy-free culture media has been described in the Dutch patent application No. 66 05 337, which is equivalent to U.S. Patent Application No. 4,51311 filed April 27, 1965 (page 4, cells 29 ff.), Recommended because these compounds in a concentration of up to 0.1% by weight & the formation of promote high cell concentrations.

According to the invention, however, a mixture of phosphates is used as a buffer in order to prevent the pH value from falling after delaying the formation of lactic acid. Obviously then the required amount is greater. One 10 times higher concentration than recommended in the last mentioned Dutch patent application, is effectively applied.

To prevent protein flocculation, which can reduce starter activity, they need Components of the culture medium do not get pasteurized after being used for inoculation with the bacteria have been brought together.

The pasteurization is otherwise usually done by heating to a temperature of 100 ° C for a Performed a period of 30 minutes ("steaming"). If a cheese whey is used, heated to 75 ° C to remove rennet and starter activity and then cooled and centrifuged is, and if this liquid is "steamed", possibly after previous addition of a Phosphate mixture or of skimmed milk (or some other protein dispersion) or of both, then occurs a coagulation of protein, which is therefore no longer available for assimilation. this leads to to a lower starter activity after the starter bacteria have been grown therein. If the same The process is carried out after the whey proteins have previously been partially digested by means of trypsin, then after the "steaming" there is actually no protein deposit; however, this method is again

expensive.

However, a satisfactory result is obtained by first "damping" and then the Centrifuged whey. After cooling to a lower temperature, the treated whey becomes with the Buffer mixture and the skimmed milk that has already been "steamed" separately.

If the buffer mixture is used as an aqueous solution, it should preferably also be pasteurized "earth. Mixing is preferably carried out in such a way that the temperature of the mixture obtained is not 65.degree exceeds. Otherwise undesirable precipitates could form, although this is not always the case.

With this separate pasteurization, no common precipitate is formed from whey and milk protein, and the activity of the starter is normal when using 5% skimmed milk. Steaming the whey is not absolutely necessary. When the whey is acidified with hydrochloric acid to a pH of 6.0 and then heated for 10 minutes to a temperature of 85 ° C and subsequently centrifuged before phosphates and skimmed milk are added, then the starter even after adding only 5% Skimmed milk has a practically normal activity.

If the bacteria have been incubated long enough after inoculating the culture medium, they can be separated from the aqueous phase, for example by means of freeze drying or by means of a Baktofuge.

It is expedient to use sodium chloride or a protein to dissolve the protein beforehand Dissolved means. If the protein is not dissolved beforehand, a baktofuge can close very quickly

will.

With the help of a Baktofuge, it is possible, when using the method according to the invention, to concentrate more than 90% of the bacteria in a culture produced according to the invention to a volume that makes up 2% of the original culture, and the concentrate is thus obtained in a continuous stream and

can process it without prior resuspension.

Said, obtained bacterial concentrate can in a known manner in a for storage and the

Transport suitable form are brought, for example by rapid freezing of the concentrate on a. Temperature of -40 ° C, preferably after adding lactose, and by further storage at one temperature preferably not above -10 ° C. The concentrated bacterial culture can also be lyophilized. The bacterial concentrate thus obtained according to the invention can simply be introduced into the milk for cheese production and it shows good activity in it. s

The bacterial concentrate obtained can also be fermented directly without prior incubation Cream for making butter can be brought in. Other fermented milk products, such as yogurt and Buttermilk, for example, can be obtained in a similar way, although, as goes without saying, the composition of the bacterial concentrate the purpose for which it is intended. must be adjusted.

Various values relating to the composition of the culture medium, its behavior on heating and the activity of the initiator obtained are given in Table A below. As far as activity is concerned, it should be noted that in order to gain an idea of the usefulness of a starter in cheese making, its total number of germs per ml is often determined. However, a better impression is obtained by measuring the rate of acid formation under precisely prescribed conditions in the so-called activity determination. For this purpose, 1% of the starter is incorporated into the cheese milk. The milk is then kept at a temperature of 30 ° C. for 6 hours, and the acid value is then determined by titration with 0.1 N NaOH solution with phenolphthalein as an indicator (Dutch standard method NEN 913). The activity is ^{expressed as 0} N; this means the number of ml of 0.1 N NaOH that are used per 100 ml of incubated mixture. This activity is indicated in the table.

Table A.

Experimental base fluid
number

Successive treatments of the basic liquid

pH then heated

humiliated during
on

10 min.

Centrifuged

30 min.

75 ⁰ C 85 ⁰ C 90 ⁰ C

IQO ⁰ C

pH with 0.04% 30 min

increases trypsin

to (BDH) 100 ⁰ C

treated heated and centrifuged

subsequently 30 min

admitted to

(Vol.%) 100 ⁰ C

Lean phosphate heated milk solution ')

Result

Before the activity seeding Toggle according to the wesender NEN ⁰ N low impact-2)

1 5 Skimmed milk 2a 6th whey b whey C. whey d whey 3a whey b whey C. whey 4a whey b whey C. whey d whey whey whey

6.0 +

4.6 +

') The phosphate solution contains per 100 ml 28.3 g Na ₂ HIPO * 2H ₂ O and 21.6 g KIl ₂ PO ₄ : ² ) No precipitate occurs:

+ formed in small quantities;
+++ there was a lot of precipitation.

6.8
6.8

yes yes yes yes

10 1 10 1 10 1 10 1 10 1 2 '/ 2 1 5 1 10 1 5 1 5

62

48 SJ
Os) 41 SJ 61 Ι — ι 63
35 ON 33 36 32 55 58 63 58 60

The invention is further illustrated by the following examples. "Wl" is a starter, the Streptococcus contains diacetilactis as a citric acid fermenting bacterium in addition to Betacoccl. »Fr8« is a starter that works as a Citric acid fermenting bacteria only contains Betacoccl.

example 1

A mixture which had been obtained from the first and second dehydration of the whey during cheese production and had already been freed as much as possible of fat and small clumps by centrifugation, was pasteurized at 90 ° C. for 10 minutes. During this period of time, more of the protein was precipitated. The precipitate was removed by centrifugation at 10 000 χ g. Together with the pretreated whey, other pasteurized liquids, such as those indicated in Table B, were placed in sterile 100 ml flasks, and culture media were prepared which were inoculated with 1% of one of the starters W1 or Fr8 . The medium was then incubated at 20 ° C. for 21 hours. Thereafter, the activity of the obtained starters in milk prepared by redissolving skimmed milk powder (low heat; L-Mllch) as well as in skimmed milk heated for 10 minutes at 115 ° C (HM !! ch), certainly. The results are given in Table B. It is clear that in experiments 3 and 5 the activity of the starter was practically as good as the activity of a starter which had been cultured in the usual way in skimmed milk (experiment 8), while in experiments 6 and 7, in which the concentration of nutrients was lowered as a result of the addition of water, the activity was lower.

In separately inoculated culture media, in some cases the lowering of the pH determined during the incubation period. The results are in Flg. 1 and 2 reproduced. Because of Due to the buffer capacity of the introduced phosphate mixture, the lowering of the pH value was initially slower than in skimmed milk, but with continued incubation it turns out that the lowering of the pH value like In a normal skimmed milk culture.

Table B.

_ * _ _ _ ι Vprcnrh

Composition of
Culture medium (in ml) attempt
1 2 73 Vi 3 4th 5 6th 7th 8th Pre-treated whey 75 - 69 Vi 69 '/ 2 46 35 23 Steamed skimmed milk - υ / 2 4th - 4th 4th 4th 75 50% aqueous phosphate
solution ') _ υ / 2 υ / 2 υ / 2 υ / 2 4% (w / v), * aqueous
Solution of sodium caseinate - _ 4th _ water - - - 23 35 46 - Starter activity 41 Wi in L milk 32 51 44 42 44 39 38 44 Wi in UHT milk 37 33 60 54 57 55 52 60 Fr8 in L milk 25 '/ 2 51 43 37 40 35 33 49 Fr8 in long-life milk 33 57 53 56 53 49 61

') 100 ml of this liquid contain: 28.3 g Na ₂ HPO ₄ · 2H ₂ O and 21.6 g KH ₂ PO ₄ ; after dilution (1:50 in the culture medium) the pH was 6.8.

* W / L = weight / volume

Example 2

A starter was made on an engineering scale. A mixture of the first and second whey dehydration, from which most of the protein had been removed by centrifugation in a sludge centrifuge after heating to 90 ° C and acidification to pH 4.6, was used, and it became a quantum of 2300 Liters of this whey brought to a temperature between 90 ° C and 95 ° C. Pumped into an incubator and cooled to a temperature of 40 ° C. with stirring and then brought to a pH of 6.8 by adding an NaOH solution. Thereafter, 120 liters of skimmed milk and an aqueous solution of 14 kg were Na ₂ HPO ₄ ■ 2H ₂ O and 11 kg KH ₂ PO ₄ in 25 kg of water, both of which had been heated to 9O ⁰ C, was added. The tank contents were then cooled to 20 ° C. and inoculated with 1% of the starter Wi. This starter was obtained from a frozen, inoculated starter culture. This is milk that has been inoculated with bacteria, not incubated, but immediately stored at freezing temperatures and thawed before use. Incubated and subsequently inoculated twice. ⁶ p

After an hour of incubation, the pH of the contents of the incubator is brought to 6.9 using NaOH, while sodium chloride up to a weight of 5 kg dissolved in 8 liters of water was introduced to give the protein to dissolve in it. The clarified starter was then centrifuged in a baktofuge and the sludge was

pumped through a cooler to a tank in which it is stored at a temperature of about 2 ° C became. A quantity of 45 l of sludge was obtained, in which a solution of 3.5 kg of lactose in 41 Water introduced '-arde. Approximately 300 grams of this liquid was placed in each of a number of cans, which were then passed through a freezer tunnel to their contents at a temperature of -35 ° C to freeze, and then they were stored at -40 ° C.

After storage for one day at -40 ° C., the total number of germs in the frozen starter concentrate was and determines its activity. After 5 days of storage at -40 ° C, the total number was determined again, to compare it to that of the Wi-Starter made according to standard procedures. In addition, the total number and the number of aroma bacteria in TGV agar (1) and '/ ·>% Wacca agar (1) (using a 3-day incubation at 25 ° C). The results are in Table C. reproduced. The activity of the starter was good, both in the freshly made and in the concentrated Starters have an approximately equal ratio between the number of aroma bacteria (Betacoccl) and the Total number was available.

(1) Th. Galesloot, F. Hassing and J. Stadhouders. NMZT.15 (1961) 127.
Table C.

Wi starter Total number
the germs (T)
(X 10 ⁷ per ml) Total number
the Betacocci (B)
(X 10 ⁶ per ml) relationship
B: T Activity ⁰ N in
L milk As a concentrate, however
Diluted 40 times after 1 day at -40 ° C 121 not determined not determined 56 after 5 days
at -40 ° C 131 33 0.025 not bestimi as fresh
Ksr-provided starter 54 15th 0.028 66

UHT milk

57

61

Example 3

5 liters of a mixture of the first and second Moikesikwäiserung in the cheese production were steamed for 15 minutes and then 10 minutes, long be! Centrifuged 10,000 χ g to remove the precipitated protein. In this pasteurized whey 5% steamed skimmed milk and 2% of a solution of 50% phosphate (prepared as in Example 1) were introduced. This mixture was then inoculated with 0.5% Wi-Starter and incubated for 18 hours at 20 ° C. This starter was then neutralized as in Example 1 and clarified with 0.1% sodium citrate, and then the bacteria were treated by centrifugation for 10 minutes! 10,000 xg removed. The sediment was suspended in part of the upper liquid up to a total volume of 1/40 of the original starter volume. This concentrate was used to make cheese from the same milk lot and according to the same process as that cheese was made with the freshly made WI starter, of which 0.8% by volume was added to the milk. A corresponding amount of 0.02 % by volume of the concentrate was used. The progressive lowering of the pH in the cheese made with the starter concentrate and in the cheese made with. freshly made starter is made in the Flg. 3 shown. After 6 weeks, the CO ₂ content of the cheeses obtained was determined. It was practically the same (0.72 and 0.76 mg / g), while the organoleptic evaluation (taste and cut) showed no differences.

Example 4

A portion of the frozen starter concentrate used in Example 2 was made into butter-making cream cultivated. No difficulties were encountered in making and the butter was from normal quality.

Example 5

Another portion of the frozen starter concentrate used in Example 2 was used to make a Ground starter used. The cheese making process went perfectly normal and the cheese showed no deviations.

Example 6

<· * In the same whey as used in Example 2, and after adding the phosphate mixture up to pasteurized to the same concentration as mentioned above, soybean pomegranate solution was used instead of Skimmed milk was added, the former being left as a 5% aqueous dispersion for 10 minutes before introduction 75 ° C had been heated. The Soyaproteln concentration obtained after mixing with the culture medium

was 0.25 and 0.50 wt. -96. After cooling to 20 ° C, the mixture was 1% of one of the starters
Vaccinated Wi or Frö. After 18 hours of incubation at 20 ° C., the activity of the starter was determined using the method
of Example 1 determined. The results are given in Table D. It was found that too
when the milk protein in the culture medium was replaced by Soyaprotsin, a starter was obtained whose
Activity was practically the same as the activity of a starter grown in skimmed milk.

Table D. Activity of Fri8 Starters (° N) in Fri8 i Culture medium L milk 47 UHT milk 64 Wi 34 Wi 57 64 67 Skimmed milk 58 61 Whey + 1% (w / v) *) Phosphate mixture 35 57 + 0.25% (w / v) Soy protein 58 61 Whey + 1% (w / v) Phosphate mixture + 0.50% soy protein

*) W / L = weight / volume

For this purpose 3 sheets of drawings

Claims (1)

Patent claims: 1. Method of preparing a mass used as inoculum for the production of starters and fermented Milk products are useful that contain at least one strain of lactose fermenting Bakiiiiennthäit and is preserved by the fact that one (a) a culture of the bacterial strain (strains) used in a culture medium containing protein breeds and, if desired, (b) subsequently in the culture medium in which the protein that is still present has possibly been converted into a dissolved form ic, the said mass constricts and (c) treating the concentrate to render it in a stable condition, thereby characterized in that a culture medium is used which (d) whey, diluted whey, low-lactose whey or an aqueous dispersion of whey powder, with a possibly too low carbohydrate content due to lactose, glucose or another ΐ5 assimilable carbohydrate can be supplemented, (e) an aqueous dispersion of skimmed milk, skimmed milk powder, soy protein, casein and / or caseinate and (0 a mixture of phosphates as a buffer mixture that has no harmful effect on the bacteria is acceptable in food from a toxicological point of view and a decrease in the pH value delayed after the formation of lactic acid, and in which the components (d) and (0 are not pasteurized together before mixing.