WO2005010170A9

WO2005010170A9 - Fermentation medium based on a highly hydrolized protein substrate

Info

Publication number: WO2005010170A9
Application number: PCT/IB2004/002361
Authority: WO
Inventors: Claudio Rottigni; Daniela Lavezzari; Salvadori Bruna Bianchi
Original assignee: Ct Sperimentale Del Latte S P; Claudio Rottigni; Daniela Lavezzari; Salvadori Bruna Bianchi
Priority date: 2003-07-25
Filing date: 2004-07-23
Publication date: 2005-03-10
Also published as: WO2005010170A3; ITMI20031528A1; WO2005010170A2; EP1656444A2

Abstract

Fermentation medium comprising a highly hydrolized protein substrate, said substrate being characterized in that it consists of low molecular weight peptides and a high percentage of free amino acids.

Description

FERMENTATION MEDIUM BASED ON A HIGHLY HYDROLYZED PROTEIN SUBSTRATE The present invention relates to a fermentation me- dium based on a highly hydrolyzed protein substrate. More specifically, it relates to a fermentation medium based on a highly hydrolyzed protein substrate and its use for the production of lactobacilli cultures, in particular Lactobacillus acidophilus, with a high cellu- lar concentration. An increasing interest has been developing over the past years in "health" food and drinks which are rightly considered by consumers as helping to obtain and maintain a better physical fitness. Among these "health" food and drinks, both fermented and non-fermented products based on milk, as well as pharmaceutical products, in which there are high concentrations of so-called probiotic microorganisms, have acquired great importance and have undergone considerable development. Among the various species of probiotic microorganisms, lactobacilli have a primary role and, in particular, Lactobacillus acidophilus _r Lactobacillus plantarum, Lactobacillus casei subsp. Casei , Lactobacillus casei subsp. Rhamnosus , Lactobacillus zeae, Lactobacillus sali- varius, Lactobacillus lactis, Lactobacillus helveticus , Lactobacillus reuteri , Lactobacillus amylovorus, Lactobacillus crispatus , Lactobacillus curvatus , Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii and all its subspecies, Lactobacillus gasseri , Lactobacillus johnsonii _r Lactobacillus delbrueckii subsp. Bul- garicus, etc. In particular, Lactobacillus acidophilus has a particularly important role among the lactobacilli listed above as it has specific beneficial effects on the human organism. As a result of the ever-growing interest in this kind of product, the demand for cultures of lactobacillus strains, in particular Lactobacillus acidophilus, both in liquid and frozen form, and above all in freeze-dried form, has increased enormously. The demand for freeze-dried cultures of these microorganisms which have a high microbial content at the production-end and which are capable of ensuring a high con- tent of live and vital cells in the product in which they are used, has particularly increased. It is not simple however to succeed in obtaining products which satisfy these requisites, above all in the case of Lactobacillus acidophilus . These microorganisms are in fact difficult to cultivate as they are particularly demanding as regards the type of nutrients they necessitate: it is in fact extremely important for there to be peptones or protein hydrolysis products as nitrogen source among the medium nutrients. Furthermore, as the interest is mainly in strains in freeze-dried form, it is essential for all the nutrients and consequently also the nitrogen source to be present in soluble form in order to obtain the maximum cellular yield in concentration. It has been surprisingly found that the use of a particular fermentation medium characterized by a certain nitrogenated source, enables cultures of lactobacilli with a high cellular concentration, to be obtained. An object of the present invention therefore relates to a fermentation medium based on a highly hydrolyzed protein substrate, said protein substrate being characterized in that it consists of low molecular weight pep- tides and a high percentage of free amino acids. In particular, 85-95% of the peptides, referring to the total quantity of peptides, which form the highly hy- drolyzed protein substrate, has a molecular weight lower than or equal to 1,000 Dalton, in soluble form. The highly hydrolyzed protein substrate consequently has 85-95% of peptides with a molecular weight lower than or equal to 1,000 Dalton, in soluble form and a percent- age of free amino acids, with respect to the total a ino acids, not lower than 45%. The fermentation medium according to the present invention based on a highly hydrolyzed protein substrate can also envisage that the protein substrate be in liquid form, in spray form, in powder or pellets. A further object of the present invention relates to the use of the fermentation medium based on a highly hydrolyzed protein substrate, for the growth of lactobacillus cultures, in particular for the growth of cultures of Lactobacillus acidophilus . The main advantage of the fermentation medium according to the present invention consists in the fact that it allows lactobacillus cultures with a high content of live and vital microbial cells, to be obtained. A further advantage of the fermentation medium according to the present invention is that the highly hydrolyzed protein substrate contains peptides in soluble form. In particular, the fermentation medium comprises 85- 95% of peptides with a molecular weight lower than or equal to 1,000 Dalton referring to the total peptides. It also contains a high percentage (45%) of amino acids in free form. The fermentation medium according to the present in- vention also comprises sugars (glucose, lactose) , yeast extracts, polysorbate and calcium carbonate. The process for the preparation of the highly hydrolyzed protein substrate according to the present invention comprises the following phases: preparation of a so- lution of the protein matrix in water; pasteurization of the solution thus obtained at a temperature ranging from 80 to 90°C; cooling of the solution to a temperature ranging from 50 to 60°C; enzymatic hydrolysis with the use of two proteolytic enzymes in sequence, the first hy- drolysis being carried out at a temperature ranging from 50 to 60°C for a time varying from 2 to 6 hours, the second hydrolysis being carried out at a temperature ranging from 50 to 60°C for a time varying from 14 to 22 hours; addition of the further components of the fermentation medium. The preparation process of the fermentation medium envisages the preparation of the highly hydrolyzed substrate as described above and with the subsequent addition of the further components which form the fermenta- tion medium. The protein substrate can be obtained starting from a protein matrix which can be of a vegetable or animal origin. The protein matrix is preferably a casein matrix. For purely illustrative purposes, the preparation process of the highly hydrolyzed protein substrate is described. In particular, the casein matrix, used as substrate for the hydrolysis, consists of a solution in spring water of a mixture made up of a concentrate of milk pro- teins and dry low-fat milk, dosed in such a quantity as to obtain a protein content ranging from 4 to 5.5% by weight; the mixture is dissolved at a temperature ranging from 30 to 40°C. The solution thus obtained is instantly pasteurized at 80°C and then cooled to 55°C. The enzymatic hydrolysis process is therefore also effected at a temperature of 55°C, using two proteolytic enzymes in sequence, maintaining the protein solution under constant vigorous stirring. Once the reaction temperature has been reached, the pH is adjusted to a value of 10.3 ± 0.1 with sodium hydroxide at 30%. 0.05% of a bacterial protease with an enzymatic activity not lower than 2.4 Anson units/g, is then added, and the mixture is left to react for three hours. After this period of time, a first aliquot is added, equal to 0.25% of a protease/peptidase with an enzymatic activity not lower than 500 amino peptidase Leucine units/g and the reaction is left to continue for 4 hours, at a constant temperature of 55°C. The second aliquot is then added, again equal to 0.25% of the same enzyme and left to react, again at 55°C for 14 hours. The highly hydrolyzed protein substrate is then ready and can be subjected to a spraying process, whereas the other components of the medium can be added. The enclosed figures illustrate various examples of hydrolysis . The determinations were effected in HPLC with the Gel Filtration technique, using a detector fixed at a wave-length of 214 nm and a column capable of selecting peptides having a molecular weight ranging from 100-7,000 Dalton. Figure 1 shows the typical hydrolysis trend. In particular, at the beginning of the hydrolysis, the non- hydrolyzed substrate has at λ = 214 nm, a percentage equal to 98.1% of components with a molecular weight ranging from 10,000 to 1,000 Dalton, with a net percentage of 69.9% with a molecular weight higher than 10,000 Dalton. Already at the end of the hydrolysis with the first enzyme, there is a shift of the peptides towards frag- ments having a lower dimension and there is a more homo- geneous weight distribution. Furthermore, the high molecular weight fragments have practically disappeared. At the end of the hydrolysis, this sample has 91% of peptides with a molecular weight lower than 1,000 Dalton. The following tables 1 and 2 indicate the composition with respect to total and free amino acids of a highly hydrolyzed protein substrate. As can be seen, the percentage of free amino acids is extremely high, as it is equal to 45% of the total amino acids present in the substrate.

Table 1 - Aminogram - Analysis in HP C on protein hydrolyzed samples The total level of free amino acids proved to be high. The_. protein hydrolyzed product prepared was highly hydrolyzed. As a percentage ratio, 45% of the amino acids produced are in free form.

Table 2 - Free ratio with respect to the -total of each single amino acid For the various amino acids, the free ratio with respect to the total proves to be extremely high. The sample prepared is highly hydrolyzed. In particular there is a percentage of well over 50% for the following amino acids: alanine, arginine, leucine, lysine, isoleucine, methionine, phenylalanine, threonine, tryptophan and va- line.

Figure 2 represents the trend of two different hy- drolyses carried out in two different fermenters in the same day, which have practically identical results. Figure 3 represents the trend of four different hy- drolyses carried out at different times in the same fermenter. For illustrative purposes, the preparation process is described of the culture medium, starting from the highly hydrolyzed protein substrate. The other components necessary for the development of the lactobacilli, are added to the highly hydrolyzed protein substrate previously obtained. In particular, lactose, glucose, yeast extract, polysorbate, CaC0₃ are added. The fermentation medium thus obtained is then pasteurized at a temperature ranging from 80 to 90°C for about 40 minutes, with the subsequent addition of anhydrous NaHC0₃ to reach a pH ranging from 5.8 to 6.4. Inoculation is then effected with the microorganism of in- terest and the incubation is effected at the optimal growth temperature of the microorganism, 37-42°C, for 7-8 hours with stirring lower than 100 rpm, the pH set-point being maintained at approximately 5 with NaOH at 30% (w/w) . At the end of the fermentation, the pH is brought to 5.80 with NaOH at 30% (w/w), anhydrous NaHC0₃ is then added to reach a pH ranging from 5.8 to 6.4, the medium is then cooled to 25°C and is left at this temperature for 15 hours, without stirring. The following day a further addition of anhydrous NaHC0₃ is effected to reach a pH ranging from 5.8 to 6.4. The following examples indicate possible uses and applications of the highly hydrolyzed protein substrate and are provided for purely illustrative and non-limiting purposes of the present invention. The percentages indicated in the examples, unless otherwise specified, are intended to be by weight. EXAMPLE 1

Preparation of a culture of Lactobacillus acidophilus with a high cellular concentration by means of a highly hydrolyzed protein, substrate in liquid form (see Figure 4 and scheme 5) .

Phase 1 — Preparation of the hydrolyzed protein product (Figure 4) . A suspension comprising a quantity of a concentrate of milk proteins and low-fat powder milk in cold water, was prepared, dosing the two powders so as to obtain percentages equal to 4.3% and 3% by weight, respectively; the mixture was dissolved at a temperature of about 35°C. The solution thus obtained was pasteurized at 80°C instantaneous and then cooled to 55°C. The enzymatic hy- drolysis process was therefore also effected at a temperature of 55°C and the protein solution was constantly maintained under vigorous stirring. As soon as the reaction temperature of _.55_°C _.had been reached, the pH was brought to a value of 10.3 ± 0.1 with sodium hydroxide at 30% (w/w). 0.5 g/1 of bacterial protease were added and the reaction was continued for three hours at 55°C. After this period, 2.5 g/1 of protease/peptidase were added and the reaction was left to proceed for 4 hours, at a constant temperature of 55°C. The addition of a second aliquot equal to 2.5 g/1 of protease/peptidase was then effected and the reaction was left to proceed, still at 55°C, for 14 hours. Phase 2 - Preparation of the mother culture (scheme 5) In correspondence with Phase 1, a strain of freeze- dried L . acidophilus is recovered, by means of re- hydration in 100 ml of MRS broth, previously sterilized at 121°C for 15 minutes. The solution, to which 0.1% of CaC0₃ and 0.1% of glucose have been added, is incubated in anaerobiosis at 37°C for 16-18 hours. A substrate, consisting of spray powder milk reconstituted at 10%, sprayed hydrolyzed protein product re- constituted at 0.5%, yeast extract reconstituted at 1% and glucose at 0.5%, is pasteurized at 90°C for 40 minutes. The microorganism is inoculated in a ratio of 5% starting from a laboratory mother culture developed at 37°C. The incubation is stopped when the pH proves to be equal to 4.25 ± 0.1.

Phase 3 - Fermentation of the substrate (scheme 5) 2% of lactose, 4%- of glucose, 3% of yeast extract, 0.1% of polysorbate and 0.2% of CaC0₃, were added to the highly hydrolyzed protein substrate obtained in Phase 1. The product is then pasteurized at 90°C for about 40 minutes, with the subsequent addition of 0.4% of anhydrous NaHC0₃ to reach a pH equal to 6.2. 5% of the mother culture (Phase 2) is added, incu- bating at 37 °C for 7 hours, with stirring lower than 100 rpm, maintaining the pH set-point at a value of 5 with NaOH at 30% (w/w) . At the end of the fermentation, the pH was brought to 5.80, again by adding NaOH at 30% (w/w) and 0.4% of anhydrous NaHC0₃ to reach a pH of 6.2. The temperature was then brought to 25°C and the tank was left for 15 hours, without stirring, at this temperature. The following day, a further addition of 0.6% of anhydrous NaHC0₃ was added. EXAMPLE 2 Preparation of a culture of Lactobacillus acidophilus with a high cellular concentration by means of a highly hydrolyzed protein substrate in sprayed form (scheme 6) . Phase 1 and 2 - phases 1 and 2 described in Example 1 are repeated. Phase 3 - Preparation of the culture medium and fermentation of the substrate. The highly hydrolyzed protein substrate obtained in Phase 1, is added, after a spraying process, in a ratio of 4% to a medium consisting of: 2% lactose, 4% glucose, 3% yeast extract, 0.1% polysorbate and 0.2% CaC0₃. The fermentation medium thus obtained is then pasteurized at 90°C for about 40 minutes, with the subsequent addition of 0.4% of anhydrous NaHC0₃, to reach a pH of 6.2. Lactobacillus acidophilus is then added to the fermentation medium, in a ratio of 5% starting from the mother culture prepared in Phase 2, incubating at 37°C for 7 hours, with stirring lower than 100 rpm, maintaining the pH set-point at a value of 5 with NaOH at 30% (w/w) . At the end of the fermentation, the pH was brought to 5.80, again by adding NaOH at 30% (w/w) and 0.4% of anhydrous NaHC0₃ to reach a pH of 6.2. The temperature was then brought to 25°C and the tank was left for 15 hours, without stirring, at this temperature. The follow- ing day, a further addition of 0.60% of anhydrous NaHC0₃ was added. EXAMPLE 3

Preparation of a culture of Lactobacillus delbrueckii subsp. bulgaricus with a high cellular concentration by means of a highly hydrolyzed protein substrate in sprayed form (scheme 7) .

Phase 1 - phase 1 described in Example 1 is repeated. Phase 2 - preparation of the mother culture. In correspondence with Phase 1, a strain of freeze- dried L , delbrueckii subsp. bulgaricus is recovered, by means of re-hydration in 100 ml of MRS broth, previously sterilized at 121°C for 15 minutes. The solution is incubated in anaerobiosis at 42 °C for 16-18 hours. A substrate, consisting of powder milk reconstituted at 10%, sprayed hydrolyzed protein product reconstituted at 0.5%, yeast extract reconstituted at 1% and glucose at 0.5%, is pasteurized at 90 °C for 40 minutes. The microorganism is inoculated in a ratio of 5% starting from a laboratory mother culture developed at 42°C. The incubation is stopped when the pH proves to be equal to 4.3 ± 0.1.

Phase 3 - Preparation of the culture medium and fermenta- tion of the substrate. A medium consisting of: 2% permeate, 4% glucose, 3% of yeast extract, 0.1% of polysorbate, 0.5% of sodium acetate and 0.2% of CaC0₃, was added in a ratio of 1% to the highly hydrolyzed protein substrate obtained in Phase 1, after the spraying process. The fermentation medium thus obtained is then pasteurized at 90°C for about 40 minutes, with the subsequent addition of 0.4% of concentrated NH₄OH to reach a pH equal to 6.2. 4% of the mother culture (Phase 2) is then added to the fermentation medium, incubating at 42°C for 7 hours, with stirring equal to 110 rpm, whereas the pH is adjusted to a value of 6.0 with concentrated NH₄OH. At the end of the fermentation, the pH was brought to 6.5, again by adding concentrated NH₄OH. The temperature was then brought to 4°C and the tank was left for 15 hours, without stirring, at this temperature. For illustrative purposes, Tables 3 and 4 compare the results of tests after fermentation, with the same time and conditions, of fermentation mediums based on a highly hydrolyzed protein substrate according to the present invention (A of Example 1 and B of Example 3) and three different mediums based on protein hydrolyzed substrates available on the market called Commerce 1, Com- merce 2 and Commerce 3. Table 3

Development of Lactobacillus acidophilus after fermentation on mediums based on different hydrolyzed broths

Table 4

Development of Lactobacillus delbrueckii subsp. bulgaricus after fermentation on mediums based on different hy- drolyzed broths

From the tables it can be observed that the highly hydrolyzed protein substrate according to the present in- vention (A and B) allows lactobacillus cultures to be obtained with a high cellular concentration (more than one/two orders of magnitude) with respect to the normal protein hydrolyzed substrates available on the market. _ The lower performance on the part of the hydrolyzed substrates Commerce 1, 2 and 3 with respect to the highly protein hydrolyzed substrate, object of the present invention, can be attributed to their lower content of free amino acids, as can be seen in Table 5.

Table 5 - Content of total and free amino acids of dif- ferent hydrolyzed products available on the market and a highly hydrolyzed protein substrate, object of the pres- ent invention.

Scheme 5 Lactobacillus acidophilus

PHASE 2 Recovery of freeze-dried strain

In 100 ml of MRS broth sterilized at 21^UC x 15'

Add: 0.1% CaCO₃ and 0.1% glucose

Incubation: 37⁰C7anaerobιosis for 16-18 hours Pre-mother: (laboratory mother)

Fermentation medium: sterilized at 110°C x 30' inoculation: 1% incubation: 37°C, aerobiosis for 16-18 hours

MOTHER

Medium

pasteurization: 90°C x 40' inoculation: 5% incubation: 37°C until pH 4.25 ± 0.1

PHASE 3 FERMENTATION:

Addition of following ingredients:

pasteurization: 90°C x 40'

Addition NaHC0₃ 0.40% inoculation T°C = 39: 5%

Incubation at: 37°C x 7 hrs

Stirring during fermentation: minimum rpm < 100

Neutralizer NaOH at 30% pH neutralization set point = 5.00 At the end of fermentation pH brought to: (with NaOH 30%) 5.8

Addition of NaHC0₃ 0.40%

Cooling overnight at +25^<>C Count at the end of fermentation MRS 5.4 |2.8 x 10^y UFC/m day after add NaHCO₃ 0.60% Count on the following day: MRS 5.4 3.0 x 10^a UFC/ml| Scheme 6 actobacillus acidophilus]

PHASE 2 Recovery of freeze-dried strain

In 100 ml of MRS broth sterilized at 121"C x 15' Add: 0.1% CaCO₃ and 0.1% glucose Incubation: 37°C, anaerobiosis for 16-18 hours

Pre-mother: ^laboratory ^ mother)

Fermentation medium: ^"sϊeπlized a Υ10°C x 30' inoculation: 1% incubation: 37°C, aerobiosis for 16-18 hours

MOTHER

Medium

PHASE 3 FERMENTATION: incubation: 37°C pH correction: in continuous SP pH = correction with: NaOH 30% Medium

Stirring: slow rpm < 100 Duration of the fermentation: 7 hrs pasteurization: 90°C x 40' pH correction : with NaHC0₃ 6.2-6.4 inoculation: 5% pH brought to: with NaOH 5.8 addition of NaHCO₃ 0.40% cooling overnight at +25°C Count at the end of fermentation MRS 5.4 1.3 x 10^& UFC/ml day after add NaHCO₃ 0.60% Count on the following day: MRS 5.4 2.2 > : 10" UFC/ml Scheme 7 \Lactobacillus delbrueckii subspec. bulgaricus

PHASE 2 Recovery of freeze-dried strain

In 100 ml of MRS broth sterilized at 2 ^υC x 15' Incubation: 42°C, anaerobiosis for 16-18 hours

Pre-mother: (laboratory mother)

MRS broth sterilized at 121 °C x 15' Inoculation: ^{~ ~ " ~}2%^" incubation: 42°C, aerobiosis for 16-18 hours

MOTHER

Medium

pasteurization: 90°C x 40' inoculation: 5% incubation: 42°C until pH 4.3 ± 0.1

PHASE 3 FERMENTATION: incubation: 42°C pH correction: in semi-continuous when pH drops to 5.00 bring it to 6.00 correction with: NH₄OH Medium

Stirring: 110 rpm only during pH correction duration of the fermentation: 7 hrs pasteurization: 90°C x 40' pH correction : with NH₄OH 6.2-6.4 inoculation 4% pH brought to: with NH₄OH 6.5 keep overnight at +4°C Count at the end of fermentation MRS 5.4 1.2 x 10⁹ UFC/ml Count on the following day: MRS 5.4 2.0 x 10° UFC/ml

Claims

1. A fermentation medium comprising a highly hydrolyzed protein substrate, said substrate being characterized in that it consists of peptides with a low molecular weight and a high percentage of free amino acids .

2. The fermentation medium according to claim 1, characterized in that the highly hydrolyzed protein substrate has 85-95% of peptides with a low molecular weight or equal to 1,000 Dalton, in soluble form and a percentage of free amino acids, with respect to the total amino acids, not lower than 45%.

3. The fermentation medium according to claim 1, characterized in that the highly hydrolyzed protein substrate is in liquid, spray, powder or pellet form.

4. The fermentation medium according to claim 1, characterized in that it comprises 85-95% of peptides with a molecular weight lower than or equal to 1,000 Dalton, referring to the total peptides.

5. The fermentation medium according to claim 1, char- acterized in that it comprises, together with the highly hydrolyzed protein substrate in liquid, spray, powder or pellet form, additional components such as lactose, glucose, yeast extract, polysorbate and/or calcium carbonate.

6. use of the fermentation medium according to any of the previous claims, as culture medium for the growth of cultures of lactobacilli.

7. Use according to claim 6, characterized in that the lactobacillus is Lactobacillus acidophilus.

8. A process , for the preparation of the fermentation medium according to one of the claims from 1 to 5, characterized in that it comprises the following phases: preparation of a solution of a protein matrix in water, the matrix having a total protein content ranging from 4 to 5,5% by weight; pasteurization of the solution thus obtained at a temperature ranging from 80 to 90°C; cooling of the solution to a temperature ranging from 50 to 60°C; enzymatic hydrolysis, at a pH ranging from 6 to 11, with the use of two proteolytic enzymes in sequence, the first hydrolysis being carried out at a temperature ranging from 50 to 60°C for a time varying from 2 to 6 hours, with a bacterial protease having an enzymatic activity not lower than 2.4 Anson units/g, the second hydrolysis being carried out at a temperature ranging from 50 to 60°C for a time varying from 14 to 22 hours with a protease/peptidase having an enzymatic activity ranging from 500 to 1,000 Leucine amino peptidase units/g; addition of the further components of the _. fermentation medium.

9. The process according to claim 8, characterized in that by the use, in the preparation of the highly hydro- lyzed substrate, of two enzymes, one with a mainly endo- peptidase activity and the other with a protease/peptidase activity.

10. The process according to claim 8, characterized in that the protein matrix can be of a vegetable or animal origin.

11. The process according to claim 8, characterized in that the protein matrix is a casein matrix.