Method for making cheese

Abstract

The present invention relates to a method for making Cheddar type and Continental type cheese with an adjunct culture comprising a Lactobacillus helveticus strain.

Claims

1. A process for improving the texture and/or taste and/or flavor of cheese, comprising: adding to milk a lactic acid bacteria culture comprising a strain belonging to a genus selected from the group consisting of Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Lactobacillus, and Enterococcus, and a non-attenuated, non-acidifying Lactobacillus helveticus strain selected from Lactobacillus helveticus strain DSM 19501 deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmBH (DSMZ) and mutants thereof, wherein the non-attenuated, non-acidifying Lactobacillus helveticus strain has at least as high cell wall bound protease activity as acidifying Lactobacillus helveticus strain DSM 19500 deposited at the DSMZ, and is not able to lower the pH more than 1.5 pH units from a starting pH of 6.5 after 10 hours incubation at 37° C. when inoculated from a fresh over-night culture at an inoculation dose of 1.5×10.sup.7 cfu/ml into 200 ml of milk, and a coagulant; and then heating the resultant mixture to a temperature in the range of 30 to 45° C.

2. The process of claim 1, further comprising, after heating the mixture to a temperature in the range of 30 to 45° C., holding the mixture at that temperature range for 5 to 70 minutes immediately before whey removal or pre-pressing under whey.

3. The process of claim 1, wherein the non-attenuated, non-acidifying Lactobacillus helveticus strain is not able to lower the pH more than 1.5 pH units from a starting pH of 6.5 after 10 hours incubation at 37° C. when inoculated from a fresh over-night culture at an inoculation dose of 1.5×10.sup.7 cfu/ml into 200 of ml milk prepared from 9.5% skim milk powder rehydrated in water and heat treated at 140° C./8 sec and 100° C./30 min.

4. The process of claim 3, wherein the non-attenuated, non-acidifying Lactobacillus helveticus strain is not able to lower the pH more than 1.3 pH units from a starting pH of 6.5 after 10 hours incubation at 37° C. when inoculated from a fresh over-night culture at inoculation dose 1.5×10.sup.7 cfu/ml into 200 ml of milk.

5. The process of claim 1, wherein the non-attenuated, non-acidifying Lactobacillus helveticus strain is the strain DSM 19501.

6. The process according to claim 1, wherein the lactic acid bacteria culture comprises a strain selected from the group consisting of Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus, Lactobacillus casei, Lactobacillus paracasei, Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus acidophilus.

7. The process of claim 1, wherein the cheese is a cheddar type cheese or a continental type cheese.

8. A cheese obtained by the process of claim 1.

9. The process of claim 1, wherein the coagulant comprises a milk clotting enzyme.

10. The process of claim 1, wherein the cheese is a low-fat cheese.

11. The cheese of claim 8, wherein the cheese is a cheddar type cheese.

12. The cheese of claim 8, wherein the cheese is a continental type cheese.

13. The cheese of claim 8, wherein the cheese is a low-fat cheese.

14. The process of claim 1, wherein the non-attenuated, non-acidifying Lactobacillus helveticus strain is a mutant of the strain DSM 19501 deposited at DSMZ, wherein the mutant has at least as high cell wall bound protease activity as the acidifying Lactobacillus helveticus strain DSM 19500 deposited at DSMZ and is not able to lower the pH more than 1.5 pH units from a starting pH of 6.5 after 10 hours incubation at 37° C. when inoculated from a fresh over-night culture at an inoculation dose of 1.5×10.sup.7 cfu/ml into 200 ml of milk.

Description

DRAWING

(1) FIG. 1 depicts the cell wall bound proteinase activity of wild type and one mutant of Lb. helveticus DSM19500. The cell wall-associated proteinase activity is shown as specific activity (Fluorescence units/OD unit), cf. the example 1.

(2) FIG. 2 depicts the acidification profile in milk of the mother stain Lactobacillus helveticus DSM19500 and Lactobacillus helveticus mutant strain DSM19501, cf. the example 2.

(3) FIG. 3 depicts the acidification profile during cheese make of control cheese, cheese made with the mother stain Lactobacillus helveticus DSM19500 as adjunct and cheese made with Lactobacillus helveticus mutant strain DSM19501 as adjunct, cf. the example 3.

(4) FIG. 4 depicts the flavour profile of control cheese, cheese made with Lactobacillus helveticus mother strain DSM19500 and cheese made with Lactobacillus helveticus mutant strain DSM19501, cf. the example 3.

DEPOSITS AND EXPERT SOLUTION

(5) The Lactobacillus helveticus mother strain was deposited 2007-07-04 at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig (DSM) and given the accession number DSM 19500. The Lactobacillus helveticus mutant strain was deposited 2007-07-04 at DSM and given the accession number DSM19501. The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

(6) The applicant requests that a sample of the deposited microorganisms stated above may only be made available to an expert, until the date on which the patent is granted.

REFERENCES

(7) Walstra et al, 2006 Dairy Science and Technology/Second edition, Taylor & Francis Cheese and Fermented Milk Foods, by Frank V. Kosikowski. El-Soda et al, 2000. Adjunct cultures: Recent Developments and Potential Significance to the Cheese Industry. Journal of Dairy Science. 83: 609-619.

(8) All references cited in this patent document are hereby incorporated herein in their entirety by reference.

EXPERIMENTAL

(9) Conventional Cheese Manufacturing

(10) Most cheese productions include a pasteurization and fat standardization of cheese milk prior to the inoculation of lactic acid bacteria starter culture (either as liquid bulk or as concentrated bacteria (frozen or freeze-dried), e.g. Chr. Hansen's Direct Vat Set). Starter culture inoculation takes typically place at temperatures from 30 to 35° C. The so-called pre-ripening time may vary between cheese types, but is typically in the range of 10 to 60 min before a coagulant is added to set the milk having a typically setting time in the range of 15 to 60 min.

(11) When the curd is formed it is cut into cubes (typically 5-20 mm) in order to facilitate syneresis of whey and concentration of milk constituents during a gentle agitation. Many cheese varieties undergo an increase of temperatures in the cheese vat (typically 36-45° C.). This is normally done with two purposes: 1) to increase the speed of syneresis and reduced the final moisture in cheese and 2) to affect the starter culture either by increasing the acidification speed of especially thermophilic LAB and/or by inducing lysis of mesophilic LAB. During the process in the cheese vat, parts of the whey may be removed and water may be added. When the curd grains obtain the right texture and acidity (typically pH 5.0 to 5.5) the grains are moulded (with or without whey removed) and pressed into shapes depending on the specific cheese variety. Most cheeses are salted, which can be done either before (typically dry salting) or after (brine-salting) the moulding. Ripening and storage conditions vary between different cheese varieties.

(12) Sensory Analysis

(13) According to International Standards (ISO 5492:1992 Sensory analysis—vocabulary) sensory perception include the attributes “taste”, “flavour”, “odour” and “aroma”. Aroma and odour are primarily associated with the perception by the olfactory organ (nose) prior to and during eating. Taste is defined as the basic tastes perceived by the taste buds in the mouth. Typically, taste is described as “sweet”, “sour/acid”, “salt” and “bitter”. Flavour is defined as a complex combination of the olfactory, gustatory (taste) and trigeminal (feeling) sensations perceived during eating. When food products are to be sensory evaluated it is custom to focus on the perception of taste and flavour.

(14) Thus, when the expression “improved/altered taste and/or flavour” is used herein, it is to be understood as the improved/altered taste and/or flavour as perceived and described by the sensory panel evaluating the cheese of the invention. This should not be taken as an exclusion of possibly altered odour and/or aroma but merely as a simple means to describe the fermented milk product of the invention.

(15) For illustration, in working example 3 herein, one suitable sensory evaluation method is the “Sensory profile”. Preferably, the test is performed according to the International Standard (ISO 13299:2003 Sensory analysis—Methodology—General guidance for establishing a sensory profile).

(16) This standard describes a guidance on the steps that are common to all sensory profiling. Sensory profiles can be established for products such as e.g. foods and beverages. Sensory profiling is based on the concept that the sensory impression made by the sample consists of a number of identifiable sensory attributes (descriptors), each of which is present to a larger or smaller degree. The list of relevant sensory descriptors, each with its intensity value, is the sensory profile. Sensory profiling can be used to compare a product/sample with a standard or with other similar products, also across all of the senses. Thus, the method is suitable for the present purpose i.e. evaluate the effect of use of an additional ingredient i.e. an adjunct culture in the production of a fermented milk product.

(17) As will be illustrated in the example 3 herein, the assessors of the sensory panel were able to compare the product made according to the present invention using a non-acidifying Lactobacillus helveticus as adjunct culture in comparison to a product made with an acidifying Lactobacillus helveticus and the product made without adjunct culture.

Example 1

Generation of Non-Acidifying Lactobacillus Helveticus Mutants and Verification of their Acidification Behaviour in Milk

(18) a) Generation of Non-Acidifying Mutants from Lactobacillus Helveticus DSM19500 by Ethyl Methane Sulphonate (EMS) Mutagenesis

(19) From frozen stock ampoule, 100 μl of strain DSM19500 were inoculated into 10 ml M17 broth+1% lactose. The culture was incubated at 37° C. (anaerobic) overnight.

(20) 150 μl Ethyl Methane sulphonate were added to the 10 ml overnight culture and the tube was sealed with para-film. The EMS-culture was incubated in a closed incubatorbox at 37° C. rotating slowly for 2 hours. 200 μl EMS-culture was inoculated into 10 ml M17 broth+1% lactose. The culture was incubated at 37° C. (anaerobic) overnight. 200 μl 87% glycerol were added to 1000 μl EMS-overnight-culture, mixed thoroughly and the stocks were stored at minus 80° C. 100 μl sample diluted to 10.sup.−4-10.sup.−7 was spread on petri-dishes with 20 ml M17 agar+1% w/v lactose+0.5 mg/ml streptomycin sulphate and 100 μl sample diluted to 10.sup.−4-10.sup.−7 was spread on petri-dishes with 20 ml M17 agar+1% lactose and incubated at 37° C., anaerobically, over night. The mutagenesis frequency was checked by counting colonies on the streptomycin plates and comparing with numbers of colonies on plates without streptomycin. The frequency of streptomycin resistant colonies should increase if the mutagenesis has worked satisfactorily. Furthermore, the number of colonies on plates without streptomycin was used for calculating the CFU/ml in order to obtain 3000 colonies when plating the mutagenized culture on Q-trays (Genetix Ltd., UK) for picking by an automated colony picker. Q-trays contained MRS agar and were incubation at 37° C. over night anaerobically. Colonies were transferred by the colony picker to microtiter plates for assessment of acidification activity in milk.
b) Assay for Assessment of Acidification Activity of Mutants in Microtiter Plates During Screening Process

(21) Milk used for acidification experiments was prepared from 9.5% skim milk powder rehydrated in water and subsequently heat treated at 140° C./8 sec and 100° C./30 min.

(22) Microtiter plates (384 wells) with milk containing 0.0476 mg/ml bromocresol purple (Na-salt) and 0.0476 mg/ml bromocresol green (Na-salt) was used for screening for mutants unable to grow in milk (method see WO2005/068982). Microtiter plates were incubated at 37° C. over night, anaerobically. If the milk changed color from blue to yellow, the strain was able to grow and made acid from lactose hence acidifying the media. If the color of milk stayed blue the strain was not able to produce acid in milk.

(23) Stability of the non-acidifying isolates was tested by re-stricking 3 times on indicator-milk agar plates: Agar plates were prepared from milk containing 0.0476 mg/ml bromocresol purple (Na-salt) and 0.0476 mg/ml bromocresol green (Na-salt) as described above. 3.375 g bacto agar were dissolved in 25 ml milliQ water and added to 200 ml of hot indicator milk. The isolates were incubated at 37° C. and it was verified that the non-acidifying isolates did not start acidifying, but stayed blue in color (pH indicator) and grew poorly on the indicator milk plates. The mother strain DSM19500 turned yellow on the indicator milk agar plates.

(24) c) Assessment of Proteinase Activity

(25) The selected isolates, unable to grow in milk, were inoculated in a 96-well microtiter plate containing MRS media. The microtiter plate was incubated at 37° C., anaerobically over night.

(26) The cell-wall associated proteinase activity was determined in a liquid handling robot as described below:

(27) Solution A: 100 mM MES buffer, pH 5.5 containing 50 mM CaCl.sub.2 (19.52 g MES and 7.35 g CaCl.sub.2. Add water to 800 ml, adjust pH to 5.5 with 1 M HCl. Fill with water to 1000 ml)

(28) Solution B: 5 mg/ml FITC-labeled casein (Sigma C3777), prepared in Milli-Q water Solution C: 5% w/v TCA (5 g TCA. Add Milli-Q water to 100 ml) Solution D: 500 mM Tris-HCl, pH 8.5 (60.55 g Tris-HCl. Add water to 800 ml, adjust pH to 8.5 with 1 M HCl. Fill with water to 1000 ml)

(29) Microtiter plates: Nunc (Product no. 167008), Nunc Black (Product no. 237105), MJR PCR plate (V-shaped, product no. HSP-9665)

(30) Tecan Liquid Handling Robot with 8- and 96-pipetting units, fluorescence/absorbance reader (Genious or Vic2), Galaxy incubator, two robotic arms, cooling units and a microtiter-plate centrifuge 1. The microtiter plate was centrifuged (2200 rpm. for 2 minutes, 6° C.) and 180 μl supernatant was asprited to waste. The cells were resuspended in 180 μl solution A (precooled to 4° C.). This washing step was repeated once more. 2. 4 μl of FITC-labeled casein (precooled to 4° C.) were dispensed to a MJR PCR microtiter plate. 3. 20 μl of cell suspension were dispensed to the MJR PCR microtiter plate. The MJR PCR microtiter plate was incubated for 3 hours or 6 hours at 37° C. (one microtiter plate per incubation time). 4. 57.5 μl of solution C were dispensed to the MJR PCR microtiter plate. The MJR PCR microtiter plate was incubated for 1 hour at room temperature. The MJR PCR microtiter plate (2200 rpm. for 2 minutes, 6° C.) was centrifuged and 45 μl were dispensed from the MJR PCR microtiter plate to a Nunc Black microtiter plate. 125 μl of solution D were aspirated to the Nunc Black microtiter plate and mixed.

(31) 5. The fluorescence was read (Genious reader) of the Nunc Black microtiter plate using excitation and emission wavelengths of 485 and 535 nm, respectively.

(32) At the same time the optical density of the washed cell suspensions is determined: 20 μl of three times washed and finally resuspended cells from step 1. were aspirated to a Nunc microtiter plate. 80 μl of solution A was added to the Nunc microtiter plate and mixed. Optical density was measured of the cell suspensions at 595 nm.

(33) FIG. 1 shows the cell wall bound proteinase activity of wild the type strain Lactobacillus helveticus DSM19500 and the mutant DSM19501. The cell wall-associated proteinase activity is shown as specific activity (Fluorescence units/OD unit). Per strain, 8 independent growing subcultures were made and standard deviations are given for these 8 replicates per strain.

(34) It was shown that the non-acidifying mutant DSM19501 has comparable cell wall bound proteinase activity than the mother strain.

Example 2

Assessment of Acidification Activity of Mutants by Direct pH Measurement in Milk

(35) As a start, working ampoules were made by incubating the strains in MRS broth at 37° C. over night. The pH was adjusted to pH 6.2 with NaOH and 20% sterilized Glycerol. The ampoules of each strain were stored frozen at −80° C. The strains were inoculated from the frozen ampoules at 1% v/v into MRS growth medium and incubated at 37° C. over night to obtain 1.5×10.sup.6 cfu/ml.

(36) 2 ml of the over night culture were inoculated in 200 ml milk and incubated at 37° C. Milk used for acidification experiments was prepared from 9.5% skim milk powder rehydrated in water and subsequently heat treated at 140° C./8 sec and 100° C./30 min. pH was measured continuously with pH probes connected to a datalogger.

(37) The mother strain acidified down to pH 4.2 at 10 hours whereas the non-acidifying strain stayed at higher pH (see FIG. 2).

Example 3

Use of Non-Acidifying Lactobacillus Helveticus Mutant DSM19501 as Adjunct Culture in the Production of Cheddar Cheese

(38) Cheddar cheeses (50% fat in dry matter and 54% moisture in non-fat substance) were made from pasteurized (72° C. for 15 s) bovine milk using chymosin (CHY-MAX™ Plus, Chr. Hansen A/S) and a frozen Cheddar-starter culture F-DVS RST-630 composed of Lactococcus lactis supsp. lactis and Streptococcus thermophilus (0.008% w/w F-DVS RST630, Chr. Hansen A/S).

(39) Experimental cheeses were made with the starter culture alone, in combination with Lactobacillus helveticus mother strain DSM19500 or in combination with the non-acidifying Lactobacillus helveticus mutant strain DSM19501 respectively. The inoculation level of the Lactobacillus helveticus strains was 6×10.sup.6 cfu/ml milk.

(40) The manufacturing method used was as previously described (conventional cheese manufacture) with a starter inoculation at 32° C., a pre-ripening time of 45 minutes and a setting time of 45 minutes. The coagulum was cut in cubes (5×5×5 mm), stirred for 15 min and then temperature increased to 40° C. in 40 minutes. Afterwards scalding agitation continued for further 20 minutes. Then the whey was drained off and the curd was further drained until pH of 5.2 5.3 was reached. The curd was milled, salted and filled into moulds. The cheeses were pressed in molds for 17 hours. One cheese of about 14 kg was obtained from each vat of 150 kg vat milk.

(41) After pressing the cheeses were removed from the moulds and vacuum packed in Cryovac® BL1L plastic bags (Cryovac, St. Neots, Belgium) and stored at 9° C. for a defined storage time.

(42) The evolution of pH values was followed during cheese make as shown below:

(43) TABLE-US-00001 Cheese with Control Cheese with adjunct cul- Time [hours] cheese adjunct cultre ture mother Step in after culture without mutant strain manufacture addition adjunct DSM19501 DSM19500 Add milk 0:00 6.62 6.62 6.62 Whey off 2:45 6.39 6.35 6.31 2. turn 3:20 5.85 5.79 5.74 3. turn 3:40 5.59 5.54 5.45 4. turn 4:05 5.39 5.36 5.24* 5. turn 4:15 5.34 5.27* 6. turn 4:30 5.29* 1 week old 5.26 5.26 5.08 cheese *milling pH reached

(44) Milling pH was reached 25 minutes earlier when the Lactobacillus helveticus mother strain DSM19500 was used as adjunct in comparison to the reference cheese without use of Lactobacillus helveticus adjunct culture.

(45) The use of the non-acidifying mutant DSM19501 had only minor influence on milk acidification, see FIG. 3.

(46) The pH at 1 week age was significantly lower in the cheese made with the Lactobacillus helveticus mother strain DSM19500 as adjunct.

(47) Sensory Evaluation

(48) An expert panel (5 panelists) evaluated the cheeses organoleptically after 10 weeks of ripening. The trays with cheese samples were tempered in a thermostatic upboard at 12° C. before the sensory evaluation. The panelists were asked to rate each cheese on a 15 cm undifferentiated scale for each sensory attribute (0 being low intensity and 15 being high intensity). The control cheese made without adjunct culture was the reference cheese for sensory profiling. A consensus sensory profile of the reference cheese was established first.

(49) Than the two cheeses containing adjunct cultures and the control cheese without adjunct culture were evaluated. A randomized three-digit identification code was given to each of the samples. Consensus profile of each cheese was established in comparison to the reference. The values given for the control cheese in the table below are averages of the first profiling as reference cheese and the second evaluation among the coded samples.

(50) Flavour profiles of Cheddar cheeses at 10 weeks age (scale 0-15 cm)

(51) TABLE-US-00002 Control cheese Cheese with ad- Cheese with ad- without adjunct junct culture junct culture culture DSM19501 DSM19500 Boiled milk 6.50 6.00 4.00 Whey 4.00 4.00 4.00 Milk fat 9.13 4.00 6.50 Sulfur 2.75 2.75 1.50 Brothy 5.50 5.25 7.75 Farmhouse 4.00 4.25 5.25 Sour 6.50 6.50 6.50 Bitter 5.25 4.00 3.50 Salt 5.63 6.50 7.75 Sweet 4.63 6.50 7.50

(52) The flavour profiles of the three cheeses (except descriptors “whey” and “sour” having the same values for all three cheeses) are shown in FIG. 4.

(53) The control cheese made without adjunct culture was related to the sensory descriptors “boiled milk”, “bitter” and “milk fat”. In contrast, the two cheeses made with adjunct Lactobacillus helveticus strains were different from the control cheese. Sensory descriptors like “salty” and “sweet” dominated more and those cheeses were described as significantly less bitter than the control cheese.

(54) It was thereby demonstrated that non-acidifying Lactobacillus helveticus strains can be successfully used as adjunct cultures to improve flavour and/or texture and/or taste in cheese.