COIC, Marie-Solenne (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
MELLEMA, Michel (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
SANDERS, Jan, Willem (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
UNILEVER PLC (a company registered in England and Wales, under company no. of Unilever House100 Victoria Embankment, London Greater London EC4Y 0DY, 41424, GB)
HINDUSTAN UNILEVER LIMITED (Hindustan Lever House, 165/166 Backbay ReclamationMaharashtra, Mumbai 0, 400 02, IN)
BECKMANN, Christoph, Hendrik (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
COIC, Marie-Solenne (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
MELLEMA, Michel (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
SANDERS, Jan, Willem (Unilever R&D Vlaardingen B.V, Olivier van Noortlaan 120, AT Vlaardingen, NL-3133, NL)
| CLAIMS 1. A process for modifying the flavour of a soy protein containing substrate, said method comprising the steps of: providing a pasteurised or sterilised aqueous liquid comprising 0.5-15% by weight of dissolved soy protein and at least 0.1% by weight carbohydrates, inoculating said soy protein-containing liquid with bacteria from a first group of mesophilic cultures selected from Lactobacillus brevis, Lactobacillus sanfranciscensis, Lactobacillus pseudomesenteroides and Lactobacillus reuteri, in an amount of between 105 to 109 CfU / ml substrate, and inoculating said soy-protein-containing liquid with bacteria from a second group of mesophilic cultures selected from Lactococcus lactis, Leuconostoc mesenteroides, Propionibacterium, Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus casei. in an amount of between 105 to 109 CfU / ml substrate, - fermenting the inoculated aqueous liquid by incubation at a temperature of 15 to 37 0C for 0.5-10 hours, wherein the bacteria selected from said first group and said second group are inoculated in ratios Cfu of first group to second group of between 10:1 and 1 :100, preferably 1 :1 to 1 :40. 2. Process according to claim 1 , wherein the Propionibacterium from the second group are selected from P. freudenreichii, P. acidipropionici, P. jensenii and P. thoenii. 3. Process according to claim 1 or 2, wherein the bacteria from a first group of mesophilic cultures comprise Lactobacillus brevis. 4. Process according to any of the preceding claims, wherein the pasteurised or sterilised aqueous liquid which is inoculated comprises soy protein in an amount of between 1 and 10% by weight, preferably between 1 and 8% by weight. 5. Process according to any of the preceding claims, wherein the pasteurised or sterilised aqueous liquid which is inoculated comprises carbohydrates in an amount of 0.1 to 10% by weight, preferably 0.2 to 5% by weight. 6. Process according to any of the preceding claims, wherein the carbohydrates comprise mono- and/or di-saccharide. 7. Process according to any of the preceding claims, wherein in total less than 6% disaccharides by weight of the fermented product are added before, during or after fermentation. 8. Process according to any of the preceding claims, wherein the fermentation is carried out at a temperature of 15 to 37°C, preferably at a temperature of between 25 and 35°C. 9. Process according to any of the preceding claims, wherein the duration of the fermentation is in the range of 1-10 hours, preferably of 2-10 hours. 10. Process according to any of the preceeding claims, wherein the process comprises a further step comprising pasteurising or sterilising the so-fermented aqueous composition. 11. Process according to any one of the preceding claims, comprising filling the fermented product into containers and subsequently sealing the filled containers, wherein edible acid is added to the fermented product prior to the filling into the containers so as to adjust the pH to less than 4.5 and optionally the fermented product is not subjected to pasteurisation or sterilisation prior, during or after the filling into the containers. 12. Process according to any one of the preceding claims, wherein the aqueous liquid containing 0.5-15 wt.% of dissolved soy protein is prepared from a soy protein source selected from the group consisting of soy isolate, soy concentrate, soy flour and combinations thereof, said soy protein source being derived from soy beans exhibiting a lipoxygenase activity of less than 15 kU/mg, more preferably of less than 10 kU/mg. 13. Process according to any of the preceding claims, wherein the substrate is a liquid, and the resulting product is a beverage. 14. Process according to any of the preceeding claims, wherein during fermentation the following changes in concentrations of flavour compounds occur: a reduction of at least one C5-C9 n-alkanal by at least 30%, preferably by at least 50% when a neutral soy milk having a protein content of 2.5 wt.% is inoculated with 107 viable cells of said culture per ml and incubated at 30°C for 4 hours, with the proviso that prior to inoculation the following aldehydes were added in the indicated amounts: 2 ppm of n- pentanal, 2 ppm of n-hexanal, 2 ppm of n-heptanal and 2 ppm of n-octanal. 15. Method according to any one of the preceding claims, wherein during the fermentation the concentration of at least one C5-Cg n-alkanal decreases by at least 30%, preferably by at least 50%. 16. Method according to any one of the preceding claims, wherein during the fermentation the concentration of (£)-2-hexenal decreases by at least 30%, preferably by at least 50%. 17. Method according to any one of the preceding claims, wherein during the fermentation the concentration of 2-methylbutanal and/or 3-methylbutanal decreases by at least 50%. |
Field of the Invention
The present invention relates to the field of fermented (or cultured) soy-containing products, especially fermented soy-based beverages, and a process to prepare such.
Background of the invention
Consumers have become more knowledgeable about protein and its role in a healthy diet. This new understanding has had a profound effect, stimulating consumer interest and demand for healthier beverages that are fortified with protein. Because beverages are a convenient way to incorporate protein into the diet, manufacturers continue to formulate new products in an effort to make protein more accessible to a wider group of consumers. The two most popular beverage proteins are milk protein and soy protein, and their various isolate derivatives. According to the U.S. Food and Drug Administration, the consumption of food products rich in soy protein can reduce cholesterol, enhance athletic performance, and even aid in the battle against diabetes. In addition, interest for milk replacement by soy protein has increased in view of, on the one hand, issues in relation to over-sensitivity and/or intolerance towards milk constituents experienced by growing numbers of consumers, a desire for products suitable for (strict) vegetarians and, on the other hand, elevated milk protein prices and supply issues that some manufacturers are experiencing relative to this commodity. Soy proteins have been proposed to replace milk proteins, either partially or totally, depending on the system, and dairy-like products have been developed based entirely on soy protein. In view of soy protein's documented health benefits it is desirable to incorporate substantial quantities of soy protein in beverages. However, incorporation of soy protein into e.g. beverages presents several challenges. The incorporation of soy protein in beverages is known to impart a noticeable aftertaste and a distinctive "beany" taste. Different types of processes to isolate soy protein have been proposed which aim to remove these undesirable (off-) flavour-notes. Unfortunately, however, it is almost impossible to completely remove the typical soy "off-notes" from sources of soy protein such as soy concentrates and soy isolates. In addition, in most product applications the intensity of soy off-notes increases during processing and storage, probably as a result of the formation of off-flavour compounds from precursor molecules. US 3,364,034 describes a method for removing characteristic flavour and/or odour from vegetable protein materials to provide a substantially bland product, comprising: inoculating said protein materials with bacteria selected from the group consisting of Lactobacillus lactis, Lactobacillus bulgaricus, Lactobacillus acidophilus, Leuconostoc citrovorum, Pediococcus cerivisiae, Pseudomonas ovalis, Pseudomonae tragi, Aerobacter aerogenes, Streptococcus lactis, incubating for 16-144 hours under conditions conducive to bacterial growth; and terminating said bacterial growth after said material is rendered substantially bland. US 4,664,919 describes a process for producing yogurt-like food, comprising fermenting soy milk with Streptococcus sojalactis. It is observed in the US patent that the yogurt-like food so obtained does not have peculiar 'green' smell of soy milk and that it has a good taste. It is further stated that the aforementioned Streptococcus strain is capable of removing the 'green' smell of soybeans and that the amount of diacetyl and acetone formed is larger than that of other lactic acid bacteria. Data provided about the Streptococcus sojalactis show that the micro-organism is capable of growing at temperatures in the range of 30-40 0 C, but not at 20 0 C or less or at temperatures of 45 0 C or higher.
US 6,599,543 relates to process for preparing a fermented soybean milk comprising: contacting dehulled and dehypocotyl whole soybeans with warm or hot water; removing warm or hot water-soluble component from the soybeans; pulverizing the soybeans to make a slurry; removing insoluble component from the slurry to make a soybean milk, inoculating a lactic acid bacterium of the genus Bifidobacterium, Lactobacillus bulgaricus and one strain selected from the group consisting of Lactobacillus acidophilus and Lactobacillus casei into the soybean milk, adding one or more saccharides which can be utilized by the lactic acid bacterium to the soybean milk, and fermenting the soybean milk to produce the fermented soybean milk. The removal of hypocotyl from the soybeans as taught by US 6,599,543 is laborious and costly.
EP-A 0 386 817 describes a process of preparing a fermented soymilk, which comprises the steps of: a) inoculating soymilk with an exocellular polysaccharide-forming lactic acid bacterium; b) incubating the inoculated soymilk; and c) recovering the fermented product.
Example 1 of the European patent application describes how 100 ml of soybean milk containing 0.5 wt.% of a added lactose was fermented with a culture of exocellular polysaccharide-forming lactic acid bacteria {Streptococcus cremoris) for 15 hours at 25 0 C, after which the pH had dropped to 4.6. After fermentation, a product was obtained with a highly viscous consistency and practically no beany flavour. EP-A 1 145 648 describes a method of preparing a lactic acid fermented soy proteinaceous food ingredient with reduced levels of flatulence inducing oligosaccharides and conserved levels of isoflavones, said method comprising: a) providing an aqueous crude soy material containing soy protein, flatulence-inducing oligosaccharides, and isoflavones; and b) simultaneously treating the aqueous crude soy material with (1 ) a source of glycosidase activity that is effective to hydrolyze the flatulence-inducing oligosaccharides to fermentable saccharides and (2) a lactic acid producing culture that ferments the fermentable saccharides. Example 2 describes how a 30% solids slurry of defatted soy flour in water was simultaneously inoculated with about 5 percent of a lactic acid culture (mixture of Lactococus lactis and Streptococcus cremoris) and about 0.01 percent of α-galactosidase enzyme. The inoculated slurry was held at 35 0 C for 4 hours, during which time the pH dropped from 6.6 to 5.3. JP-A-2004-261003 describes a method of preparing fermented soybean milk by fermenting soybean milk and by adding palatinose (isomaltulose) before, during or after fermentation. In the Japanese application it is observed that the grass smell inherent to soybean milk can be reduced to a certain extent by fermenting soybean milk with a combination of a lactic acid bacterium and a bifidobacterium, but that satisfactory results cannot always be established, in particular due to the taste and smell of acetic acid, which is metabolic product of the fermentation. Palatinose is incorporated in the fermented soybean milk to suppress the unpleasant taste, fermentation smell, harshness and acetic acid smell generated in the course of lactic acid fermentation or acetic acid fermentation. The examples of the Japanese patent application describe the preparation of fermented drink yogurts from a soybean milk to which isomaltulose and sucrose are added prior and/or after fermentation. In the examples a starter culture is used that comprises Lactobacillus delbrueckii subsp. Bulgaricus and Streptoccus thermophilus. The drink yogurts disclosed in the Japanese application are extremely sweet as they contain more than 8 wt.% of added disaccharide (isomaltulose and/or sucrose). Murti TW et al., Journal of Food Science (1993), 58(1 ), pages 153-157 describe experiments in which soymilk and cow milk were inoculated with Streptococci, Lactobacilli in the absence or presence of Bifidobacteria and wherein the concentration of a number of volatile compounds was monitored during fermentation. The results show that during fermentation of soy milk at 42 0 C the concentration of n-hexanal was decreased from 2.31 ppm to 0.55 ppm (no Bifidobacteria) or 0.45 ppm (with Bifidobacteria) after 4 hours. The lactic acid content after 4 hours of fermentation was at least 0.4 wt.%.
There is a desire for a soy protein-containing food product which is processed such that it has a reduced amount of undesired flavour components (compared to products not so processed), and preferably such process should be able to deliver products (depending on the conditions) that can have a flavour character in the range of bland to slightly dairy (but not a real dairy character). This also means that it is desired that the pH of the fermented ptoduct does not drop below 6. Such more bland character would allow processing into various other products. Preferably, such should be achievable with a limited number of different ingredients, to allow for convenient processing.
Summary of the Invention
It has now been found that the above objective can be achieved, at least in part, by a process for modifying the flavour of a soy protein containing substrate, said method comprising the steps of: providing a pasteurised or sterilised aqueous liquid comprising 0.5-15% by weight of dissolved soy protein and at least 0.1 % by weight carbohydrates, inoculating said soy protein-containing liquid with bacteria from a first group of mesophilic cultures selected from Lactobacillus brevis, Lactobacillus sanfranciscensis,
Lactobacillus pseudomesenteroides and Lactobacillus reuteri, (flavour removing bacteria) in an amount of between 10 5 to 10 9 CfU / ml substrate, and inoculating said soy-protein-containing liquid with bacteria from a second group of mesophilic cultures selected from Lactococcus lactis, Leuconostoc mesenteroides, Propionibacterium, Lactobacillus paracasei, Lactobacillus fermentum,
Lactobacillus plantarum and Lactobacillus casei, (flavour-forming bacteria) in an amount of between 10 5 to 10 9 CfU / ml substrate,
- fermenting the inoculated aqueous liquid by incubation at a temperature of 15 to 37 0 C for 0.5-10 hours, wherein the bacteria selected from said first group and said second group are inoculated in ratios Cfu of first group to second group of between 10:1 and 1 :100, preferably 1 :1 to 1 :40. Detailed Description of the Invention
It is known from literature that when soy protein-containing substrates are fermented with thermophilic lactic acid bacteria, such thermophilic LAB's may be able to provide beneficial flavour components to such substrate. It was now surprisingly found, however, that specific mesophilic bacteria can also do that. This group of "flavour-generating" bacteria herein comprises lactic acid bateria Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus casei, as well as the bacterium Propionibacterium.
It was also found that other specific mesophilic (lactic acid) bacteria can decrease the level of certain components which are responsible for the typical soy off-tastes. This group of "flavour- removing" lactic acid bacteria herein comprises Lactobacillus brevis, Lactobacillus sanfranciscensis, Lactobacillus pseudomesenteroides and Lactobacillus reuteri. Additionally, it was found that it is possible that one can carry out fermentations with bacteria from both mesophilic groups at the same time, provided (to achieve the benefits as aimed for) that one ensures inoculation is carried out such that one achieves a certain ratio of bacteria in both groups.
It was also surprisingly found that depending on the reaction conditions (e.g. fermentation time) one could produce a product having a more dairy character in taste, or a more bland character, and for both of which the level of off-flavour components is reduced. This brings the benefit of being able to produce a variety of products with different taste profiles, whilst only two different mesophilic bacteria strains are used, which is definitely an advantage form a processing point of view, as it gives flexibility in manufacturing with a limited number of bacteria strains which one needs to keep.
In the process according to the invention, the Propionibacterium from the "flavour-generating" group are preferably selected from P. freudenreichii, P. acidipropionici, P. jensenii and P. thoenii. Also, in the process according to the present invention, the bacteria from a first ("flavour- removing") group of mesophilic cultures preferably comprise Lactobacillus brevis. The term "lactic acid bacteria" as used herein refers to acid tolerant, non-sporulating, non- respiring rod-shaped Gram positive bacilli or cocci that produce lactic acid as the major metabolic endproduct of carbohydrate fermentation. As used herein, the term "lactic acid bacterium" does not encompass Bifidobacterium or Propionibacterium.
Although the process as set out herein can be employed a a wide degree of protein levels (e.g. 0.5-15%) in the substrate, it is preferred that the pasteurised or sterilised aqueous liquid which is inoculated comprises soy protein in an amount of between 1 and 10% by weight, preferably between 1 and 8% by weight. "Soy protein content", as used herein, refers to the total amount of soy protein and soy protein derived peptides contained in the fermented product. The fermented product may be based on soy protein, on soy protein hydrolysate or combinations thereof. As will be understood by the skilled person (enzymatic) hydrolysis of peptide bonds may occur during fermentation. The substrate that is fermented in the present method, i.e. the aqueous liquid containing 0.5- 15 wt. % of dissolved soy protein is preferably prepared from a soy protein source selected from the group consisting of soy isolate, soy concentrate, soy flour and combinations thereof. According to a particularly preferred embodiment, the latter soy protein sources were obtained from soy beans exhibiting very low lipoxygenase activity, e.g. a lipoxygenase activity of less than 15 kU/mg. Even more preferably the soy protein source is derived from soy beans exhibiting a lipoxygenase activity of less than 10 kU/mg, most preferably of less than 8 kU/mg. Likewise, it is advantageous to prepare the present aqueous liquid from a soy protein source having a lipoxygenase activity of less than 5 kU per gram of soy protein. Most preferably the soy protein source has a lipoxygenase activity of less than 1 kU per gram of soy protein. Lipoxygenase activity is suitably determined spectrophotometrically by using a dye-coupling assay specific for hydroperoxides generated from linoleic acid, as fully described by Anthon and Barrett (J. Agric. Food Chem. 2001, 49, 32-37).
The present method preferably employs soy protein derived from soybeans that have not been dehypocotylized (as such avoids additional process steps). Accordingly, the aforementioned soy isolate, soy concentrate and/or soy flour are advantageously derived from optionally dehulled soybeans that still comprise the hypocotyl. Most preferably the latter sources of soy protein sources are derived from dehulled soybeans that still comprise the hypocotyls. The term "dehypocotylized soybeans" as used herein refers to soybeans from which the hypocotyl has been removed. Hypocotyl is a botanical term for a part of a germinating seedling of a seed plant. As the plant embryo grows at germination, it sends out a shoot called a radicle that becomes the primary root and penetrates down into the soil. After emergence of the radicle, the hypocotyl emerges and lifts the growing tip above the ground, bearing the embryonic leaves (called cotyledons) and the plumule that gives rise to the first true leaves. The hypocotyl is the primary organ of extension of the young plant and develops into the stem.
For good growth of the mesophilic bacteria it is preferred that some carbohydrates are present in the substrate. Such can be added as such, but are frequently already part of a soy protein-containing preparation which is used as a substrate. Hence, it is preferred that the pasteurised or sterilised aqueous liquid which is inoculated comprises carbohydrates in an amount of 0.1 to 10% by weight, preferably 0.2 to 5% by weight. Such carbohydrates are preferably simple carbohydrates such as mono- and/or di-saccharide. Preferred carbohydrates are in this connection glucose, fructose, galactose, sucrose, raffinose, stachyose, lactose and combinations thereof. However, the present method enables the preparation of a pleasant tasting beverage without using high amounts of sweetener to mask soy off-flavour notes. Thus, advantageously less than 6 wt.% of disaccharides are added before, during or after fermentation. According to a particularly preferred embodiment, the fermented product in the sealed container contains less than 5 wt.% of disaccharides, most preferably less than 4 wt.% of disaccharides. According to yet another preferred embodiment, the packaged fermented product contains less than 8 wt.% of mono- and/or disaccharides, most preferably less than 5 wt.% of mono- and/or disaccharides.
The fermentation is preferably carried out at a temperature for which the bacteria from the two strains used work optimal. In this connection, it is an advantage that they are both mesophilic.
Following this, it is preferred that the fermentation is carried out at a temperature of 15 to 37°C, preferably at a temperature of between 25 and 35°C.
The duration of the fermentation herein typically lies in the range of 1-10 hours, more preferably of 2-10 hours. According to another preferred embodiment the fermentation time does not exceed 14 hours, even more preferably it does not exceed 10 hours and most preferably it does not exceed 8 hours. For manufacturing a product which can be sold commercially, it is preferred that the process comprises a further step comprising pasteurising or sterilising the so-fermented aqueous composition.
The process according to the present invention may also comprise the steps of filling the fermented product into containers and subsequently sealing the filled containers, wherein edible acid is added to the fermented product prior to the filling into the containers so as to adjust the pH to less than 4.5 and optionally the fermented product is not subjected to pasteurisation or sterilisation prior, during or after the filling into the containers. In this, the edible acid is added to the fermented product before the fermentation has reached the point at which product inhibition prevents further production of lactic acid by lactic acid bacteria.
Although the resulting product may be a liquid or e.g. a set-yoghurt-type product, the present method is advantageously employed to produce a fermented aqueous liquid that can be used as a base for the production of a beverage. In accordance with this particular embodiment of the invention the fermented product obtained in the present method has a relatively low viscosity, e.g. a viscosity at a temperature of 7 0 C of less than 50 mPa.s at 100 s "1 , most preferably of less than 25 mPa.s at 100 s ' \ A viscosity of 50 mPa.s at 100 s "1 means that the product is 50 times more viscous than water and about 25 times more viscous than milk. Viscosity can suitably be measured with the help of a rheometer AR1000 (TA Instruments, Etten-Leur, the Netherlands), using a 40-mm diameter, 2% angle cone measuring system. A steady-state shear process should be used, increasing shear rate from 0.01 to 250/s. The measuring temperature is 7 0 C and only the data point at 100 s "1 is used.
As mentioned earlier, the presently claimed process is capable of reducing the level of certain components present in soy protein-containing preparation, which components are responsible for part of the perceived off flavour of many soy preparations. C 5 -C 9 n-alkanals and (E)-2- hexenal are flavour molecules that are believed to be largely responsible for the 'beany' off- flavour often found in soy based products. The inventors have discovered that it is possible to remove typical soy-related off-notes in a very effective way using the process as set out herein. In this connection, it is preferred that during fermentation the following changes in concentrations of flavour compounds occur: a reduction of at least one C 5 -C 9 n-alkanal by at least 30%, preferably by at least 50% when a neutral soy milk having a protein content of 2.5 wt. % is inoculated with 10 7 viable cells of said culture per ml and incubated at 3O 0 C for 4 hours, with the proviso that prior to inoculation the following aldehydes were added in the indicated amounts: 2 ppm of n-pentanal, 2 ppm of n-hexanal, 2 ppm of n-heptanal and 2 ppm of n-octanal.
In this connection, it is also preferred that during the fermentation the concentration of at least one C 5 -Cg n-alkanal decreases by at least 30%, preferably by at least 50%. Likewise, it is preferred that during the fermentation the concentration of (£)-2-hexenal decreases by at least 30%, preferably by at least 50%. And similarly, it is preferred that during the fermentation the concentration of 2-methylbutanal and/or 3-methylbutanal decreases by at least 50%.
The invention is further illustrated by means of the following non-limiting examples:
EXAMPLES
Analytical methods
Analysis of off-flavour volatiles and diacetyl by Solid Phase Micro Extraction (SPME) followed by GC-MS
2 g of sample were put in a 20 ml headspace vial and sealed with an airtight cap.
Samples were analyzed by means of solid phase micro extraction. Fiber used: Carboxen/PDMS 85 μm ex. Supelco.
Analyses were carried out on an Agilent GC/MS (MS: LECO Pegasus IV TOF; 15 scans/s; m/z: 29-250 at 1700 V), equipped with a Gerstel CIS-4 injector and a Gerstel MPS-2 autosampler with SPME option. Column: VF-5; 50m * 0.2 mm * 0.33 μm
GC program:
• 40°C(2 min) -(37min)->16O 0 C(O min)-(207min)->250°C (2 min)
• Gas: Helium
• Flow: 1 ml/min, constant flow
SPME Sampling time: 35 min at 40 0 C Desorption: 40 minutes at 170 0 C Split-less time: 2 min. Sensory analysis
The fermented products and the non-fermented soy base were evaluated/tasted/smelled by an expert tasting panel (n=15), with a ranking test on soy and dairy/yoghurt intensity or a descriptive test by scoring on an unstructured scale from 1 to 10 on soy and dairy-related descriptors (e.g. milk, buttery).
Lipoxygenase activity
Lipoxygenase activity was determined spectrophotometrically by using a dye-coupling assay specific for hydroperoxides generated from linoleic acid, as fully described by Anthon and Barrett {J. Agric. Food Chem. 2001 , 49, 32-37).
Enzyme extracts were obtained by homogenising 100 mg of defatted soybean grindings in 20 ml of 100 mM pH 6.0 Na 2 HPO 4 buffer + 1 % w/v NaCI then centrifuging at 15,000 rpm at 4°C for 30 minutes and filtering the supernatant through a 0.2 μm filter.
To 500 μl of a solution of 10 mM 3-(dimethylamino)benzoic acid in 100 mM pH 6.0 Na 2 HPO 4 was added 20 μl of 27 mM linoleic acid dispersed in 1.4% w/v Tween 20, and 10 μl of enzyme extract. After 5 minutes, 500 μl of a solution containing 0.2 mM 3-methyl-2-benzothiazolinone hydrazone and 0.1 mg/ml bovine haemoglobin was added. After a further 5 minutes, 500 μl of 1 % w/v sodium lauryl sulphate was added to terminate the reaction. The absorbance at 598 nm was then measured. The above actions can be carried out in a single 4.5 ml 1 cm path cuvette.
A standard curve was produced by reacting dilutions of soybean lipoxygenase from Sigma- Aldrich, of certificated activity. This was used to calculate the activities of the extracts from the soybeans. Blank readings were subtracted, obtained using denatured enzyme extracts heated at 95°C for 30 minutes. One unit of activity is the increase of 0.001 absorbance units at 598 nm per minute from the hydroperoxidation of linoleic acid.
Viable counts The number of live bacteria in the cultures was determined by plate counting appropriate dilutions of samples containing Lactobacillus brevis and Lactobacillus fermentum on MRS agar and anaerobic incubation for 3 days at 30 0 C. Numbers are expressed as Colony forming units per ml of product (Cfu/ml). Strains
Lactobacillus brevis Lb20 (CBS122084, deposited under the Budapest treaty at Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) Lactobacillus fermentum LMG 8154 (freely available from BCCM/LMG, for info: http://bccm.belspo.be/about/lnna . php)
Lactobacillus casei 431 (commercial culture of available under this name from Chr Hansen, Denmark)
Example 1 Combination of Lactobacillus brevis & Lactobacillus fermentum at different ratios.
A: raw material
A soy base was prepared by dissolving 5.5% Sunopta SSFR powder (resulting in a protein concentration of 2.5% protein) and 3% sucrose in water. This mix was preserved by a heat treatment for 36 seconds at 79-85 0 C, packed aseptically and cooled for further storage at 5 0 C.
B: fermentation
Precultures of Lactobacillus brevis Lb20 (CBS122084, deposited under the Budapest treaty at Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) and Lactobacillus fermentum LMG 8154 (freely available from BCCM/LMG, for info: http://bccm.belspo.be/about/lmg.php) each were prepared by overnight cultivation in MRS broth at 30 0 C. Precultures were washed with water and concentrated 5-10 folds. Cell density of the concentrated preculture was measured by Optical Density (OD) at 600nm. For Lactobacillus brevis Lb20, OD was adjusted to 2.5, and for Lactobacillus fermentum LMG 8154, suspensions of OD 2.5, 5 and12.5 were prepared.
Soy base was inoculated with 2% of each of the single cultures or with 1% L. brevis OD 2.5 and 1% L. fermentum with diverse OD, as set out in table 1 below. Experiments covered inoculation amount ratio in OD of L. brevis: L. fermentum from 1 :1 , 1 :2 and 1 :5. These values in OD correspond to ratio's expressed in Cfu (colony forming units) to: 3:1 , 2:1 , and 1 :2, respectively.
After inoculation with single cultures and with combinations of cultures as set out in table 1 below, the soybases were incubated at 3O 0 C. Samples were taken for volatile measurement and the pH of the soy base was checked at regular intervals (Table 1 ). After 3 hour incubation, samples were pasteurized at 85 0 C for 30 minutes and cooled before sensory test. Inoculation of soy base with 2% of a cell suspension with OD 2.5 reflected a cell count of 2.0x10 7 Cfu/ml for L. brevis and 6.0x10 6 Cfu/ml for L. fermentum.
Table 1 Culture composition and pH of time course of soy fermentation with different cultures.
The pH measurements show that L. fermentum alone acidified the soy base more effectively than L brevis. Mixed cultures of L. brevis and L fermentum achieved acidification to pH 6.0 within 6h and increasing the ratio of L. fermentum led to more acidic product, as seen by product HZ251 108-B1 F5.
C: volatiles
An aliquot of the sample was subjected to SPME followed by GC-MS analysis. It was found that during fermentation with combined cultures, the peak areas for diacetyl (contributing to the positive flavour) and hexanal (contributing to the off-flavour) had changed in absolute numbers, but not quantitatively, as shown in Table 2. Table 2 Diacetyl and hexanal content of soy samples fermented with different amounts of L. brevis and L. fermentum as a % in Peak area of the maximum in the experiment
Thus a combination of Lbrevis : Lfermentum in a ratio of at least 1 :5 (OD) is required to achieve the same amount of diacetyl production as with Lfermentum only while at the same time the off-flavour removal effect of L. brevis is kept. D: sensory
The soybases inoculated with a combination of Lactobacillus brevis and Lactobacillus fermentum in ratios 1 :1 and 1 :2 (in OD) were not significantly different from the non- fermented product. Differences were found between the products but were not statistically significant based on the number of panelists.
The soybase inoculated with Lactobacillus fermentum and the the soybase inoculated with a combination of Lactobacillus brevis and Lactobacillus fermentum in ratio 1 :5 (in OD) were significantly lower in soy perception than the non-fermented product and the soybase product inoculated with Lactobacillus brevis and was as well the only product which was evaluated as being significantly higher in Yoghurt / Dairy taste than the non-fermented product and the soybase product inoculated with Lactobacillus brevis only.
Example 2
Combination of Lactobacillus brevis & Lactococcus lactis at different ratios.
A: raw material
A soy base was prepared by dissolving 5.5% Sunopta SSFR powder (resulting in a protein concentration of 2.5% protein) and 3% sucrose in water. This mix was preserved by a heat treatment for 36 seconds at 79-85 0 C, packed aseptically and cooled for further storage at 5 0 C.
B: fermentation
Precultures of Lactobacillus brevis Lb20 (CBS122084, deposited under the Budapest treaty at
Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) and Lactococcus lactis ssp lactis var. diacetylactis SD803 (used in EP 483888-B1 ) each were prepared by overnight cultivation in MRS broth and M17 broth, respectively, at 30 0 C. Precultures were washed with water and concentrated 5-10 folds. Cell density of the concentrated preculture was measured by Optical Density (OD) at 600nm. For Lactobacillus brevis Lb20, OD was adjusted to 2.5, and for Lactococcus lactis ssp lactis var. diacetylactis SD803, suspensions of OD 2.5, 12.5, 25 and 37.5 were prepared.
Soy base was inoculated with 2% of each of the single cultures or with 1% L. brevis OD 2.5 and 1% L. lactis with diverse OD, as set out in table 3 below. Experiments covered inoculation amount ratio in OD of L. brevis: L. lactis from 1 :5, 1 :10 and 1 :15. These values in OD correspond to ratio's expressed in Cfu (colony forming units) to 1 :4, 1 :8, and 1 :12, respectively. After inoculation with single cultures and with combinations of cultures as set out in table 3 below, the soybases were incubated at 3O 0 C. Samples were taken for volatile measurement and the pH of the soy base was checked at regular intervals (Table 3). After 3 hour incubation, samples were pasteurized at 85 0 C for 30 minutes and cooled before sensory test. Inoculation of soy base with 2% of a cell suspension with OD 2.5 reflected a cell count of 2.9x10 7 Cfu/ml for L. brevis and 2.3x10 7 Cfu/ml for L. lactis.
Table 3 Culture composition and pH of time course of soy fermentation with different cultures.
The pH measurements show that L. lactis alone acidified the soy base more effectively than L brevis. Mixed cultures of L.brevis and L. lactis achieved acidification to pH 6.0 within 6h and increasing the ratio of L. lactis led to more acidic product, as seen by product B1 L15.
C: volatiles
An aliquot of the sample was subjected to SPME followed by GC-MS analysis. It was found that during fermentation with combined cultures, the peak areas for diacetyl (contributing to the positive flavour) and hexanal (contributing to the off-flavour) had changed in absolute numbers, but not quantitatively, as shown in Table 4. Table 4 Diacetyl and hexanal content of soy samples fermented with different amounts of L. brevis and L. lactis as a % in Peak area of the maximum in the experiment
Thus a combination of L. brevis : L. lactis in a ratio of at least 1 :5 (OD) is required to achieve the maximum amount of diacetyl production while at the same time the off-flavour removal effect of L. brevis is kept.
D: sensory The soybase inoculated with a combination of Lactobacillus brevis and Lactobacillus lactis in ratios 1 :5, 1 :10 and 1 :15 (in OD) were lower in soy perception than the non-fermented product and the soybase product inoculated with Lactobacoccus lactis only. All combinations were as well evaluated as being significantly higher in Milk / Buttery taste than the non- fermented product and the soybase product inoculated with Lactobacillus brevis only. These conclusions are well aligned with the Volatiles analyses.
Example 3
Combination of Lactobacillus brevis & Lactobacillus casei at different ratios.
A: raw material
A soy base was prepared by dissolving 5.5% Sunopta SSFR powder (resulting in a protein concentration of 2.5% protein) and 3% sucrose in water. This mix was preserved by a heat treatment for 36 seconds at 79-85 0 C, packed aseptically and cooled for further storage at 5 0 C.
B: fermentation Precultures of Lactobacillus brevis Lb20 (CBS122084, deposited under the Budapest treaty at Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) and Lactobacillus casei 431 (freely available from Chr Hansen under this trademarked name) each were prepared by overnight cultivation in MRS broth at 30 0 C. Precultures were washed with water and concentrated 5-10 folds. Cell density of the concentrated preculture was measured by Optical Density (OD) at 600nm. For Lactobacillus brevis Lb20, OD was adjusted to 2.5, and for Lactobacillus casei 431 , suspensions of OD 2.5, 12.5, and 25 were prepared. Soy base was inoculated with 2% of each of the single cultures or with 1% L. brevis OD 2.5 and 1% L. casei with diverse OD, as set out in table 5 below. Experiments covered inoculation amount ratio in OD of L. brevis: L. casei from 1 :1 , 1 :5 and 1 :10. These values in OD correspond to ratio's expressed in Cfu (colony forming units) to 1 :2, 1 :8, and 1 :15, respectively.
After inoculation with single cultures and with combinations of cultures as set out in table 5 below, the soybases were incubated at 3O 0 C. Samples were taken for volatile measurement and the pH of the soy base was checked at regular intervals (Table 5). After 3 hour incubation, samples were pasteurized at 85 0 C for 30 minutes and cooled before sensory test. Inoculation of soy base with 2% of a cell suspension with OD 2.5 reflected a cell count of 2.9x10 7 Cfu/ml for L. brevis and 4.4x10 7 Cfu/ml for L. casei. Table 5 Culture composition and pH of time course of soy fermentation with different cultures.
The pH measurements show that L casei alone acidified the soy base more effectively than L brevis. Mixed cultures of L.brevis and L. casei achieved acidification to pH 6.0 within 6h and increasing the ratio of L casei led to more acidic product, as seen by product FB060609- B1 LC10.
C: volatiles An aliquot of the sample was subjected to SPME followed by GC-MS analysis. It was found that during fermentation with combined cultures, the peak areas for diacetyl (contributing to the positive flavour) and hexanal (contributing to the off-flavour) had changed in absolute numbers, but not quantitatively, as shown in Table 6.
Table 6 Diacetyl and hexanal content of soy samples fermented with different amounts of L brevis and L. casei as a % in Peak area of the maximum in the experiment
Thus a combination of L. brevis : L.casei in a ratio of at least 1 :10 (OD) is required to achieve a significant production of diacetyl while at the same time the off-flavour removal effect of L. brevis is kept. D: sensory
The soybase inoculated with a combination of Lactobacillus brevis and Lactobacillus casei in ratio 1 :10 (in OD) was evaluated as being significantly higher in Milk / Buttery taste than the non-fermented product and the soybase product inoculated with Lactobacillus brevis only. It was as well evaluated as and lower in soy off-flavour than the non-fermented sample. These conclusions are well aligned with the Volatiles analyses.
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