Lactobacillus suantsaii strain and use thereof

Abstract

The invention relates to a Lactobacillus suantsaii strain and the use thereof. The Lactobacillus suantsaii strain may be used for the production of e.g. a food product, preferably a food product with decreased salt content, highly preferably a pepper condiment with decreased salt content.

Description

[0001] Lactobacillus suantsaii strain and use thereof

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a Lactobacillus suantsaii strain and the use thereof. The Lactobacillus suantsaii strain may be used for the production of e.g. a food product, preferably a food product with decreased salt content, highly preferably a pepper condiment with decreased salt content.

[0004] BACKGROUND OF THE INVENTION

[0005] Traditional Hungarian pepper paste condiments are non-fermented, chemically preserved products (Hagyomanyok - Izek - Regiok. Magyarorszaghagyomanyos es tajjellegu mezogazda- sagi es elelmiszeripari termekeinek gyujtemenye. Volume I. Pages 57-58. FVM AMC Kht. ISBN 963 212 953 9). Until the 1960s, such products were only made at home, but since then they have also appeared as industrial products and spread to the majority of households. Today, pepper condiment has become an export product with a growing turnover, which requires proper preservation and protection against microorganisms. The preservation process - which inhibits the growth of both pathogenic and spoilage microorganisms - of pepper condiment rests on 3 main pillars:

[0006] • pH reduction by adding an acidity -regulating additive - primarily, but not exclusively, citric acid - to prevent the growth of pathogenic microorganisms or toxin production;

[0007] • reduction of water activity by adding table salt (>10% by weight, typically in an average concentration of 11.0-11.5% by weight);

[0008] • adding a preservative (potassium sorbate, sometimes sodium benzoate) in order to inhibit the growth of certain yeast / fungi or bacteria.

[0009] A large amount of salt is therefore typically added to the pepper paste made from raw ground pepper (paprika).

[0010] Similar pepper flavoring products exist in other parts of the world as well and are widely used, such as "Sambal Oelek", however, they have a much lower pH (<3.5, but in some cases ~3, 0) and have a more sour taste due to either the larger amount of acidity regulator agents (e.g. acetic acid, vinegar, citric acid etc.) or to the conversion of the majority of the fermentable sugars into lactic acid, so their use for flavoring food is much more limited than that of traditional Hungarian pepper flavorings.

[0011] The broad range of use of traditional Hungarian pepper flavorings is ensured by the fact that unlike vegetable products produced with lactic acid fermentation or with the addition of vinegar (acetic acid), they are not overly acidified (pH>3.6-3.8). However, the high salt content of the products is in contrast with today's nutritional trends. Therefore, there is a need for a preservation method and a pepper condiment produced with it, which provides protection similar to that of traditional products against pathogens or against spoilage-causing microorganisms, and at the same time it has a lower salt content, so that the amount of the acidity-regulating substance(s) added needs not to be increased and the pH to be decreased.

[0012] SHORT DESCRIPTION OF THE INVENTION

[0013] We have unexpectedly found that the salt content of pepper condiments may be descreased while avoiding excessive acidification, and a product with an appropriate shelf-life may be produced by the use of Lactobacillus suantsaii bacteria.

[0014] The invention provides the use of Lactobacillus suantsaii bacteria for the production of a pepper condiment, and also provides bacteria belonging to L. suantsaii strain UNI 1801 and bacteria derived thereof suitable for the same. The invention provides bacteria of Lactobacillus suantsaii (Levilactobacillus suantsaii) strain UNI 1801. The L. suantsaii strain UNI 1801 was deposited according to the Budapest Treaty with Mezogazdasagi es Ipari Mikroorganizmusok Nemzeti Gyujtemenye (National Collection of Agricultural and Industrial Microorganisms; Hungary, 1118 Budapest, Somloi road 14-16.) on 7 March 2022. The accession number is NCAIM (P) B 001503.

[0015] The invention also provides bacteria derived (e.g. by culturing or modifying bacteria belonging to L. suantsaii strain UNI 1801) from L. suantsaii strain UNI 1801 (bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801), which have a NaCl-tolerance of at least about 5% (w / w), at least about 6% (w / w), at least about 7% (w / w), at least about 8% (w / w), at least about 9% (w / w), at least about 10% (w / w) or at least about 11% (w / w), preferably at least about 11%.

[0016] Preferably the bacteria derived from L. suantsaii strain UNI 1801 are capable of fermenting a carbohydrate present in pepper. Preferably the pH of the pepper fermented by the bacteria derived from L. suantsaii strain UNI 1801 does not decrease below about pH=3.4, preferably below about pH=3.5, preferably below about pH=3.6-4.0, highly preferably about pH=3.6, highly preferably about pH=3.8, preferably about pH=4.0. Preferably fermentation in pepper by the bacteria derived from L. suantsaii strain UNI 1801 stops at a pH of about 3.4-4, highly preferably at a pH of about 3.6-4.0. Preferably the bacteria derived from L. suantsaii strain UNI 1801 are capable of metabolizing citric acid, glucose and / or fructose. Preferably the bacteria derived from L. suantsaii strain UNI 1801 are capable of producing an acid from one or more of the following substrates: L- arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannitol, N-acetyl glucosamine, arbutin, esculin, potassium gluconate. Preferably the bacteria derived from L. suantsaii strain UNI 1801 are capable of producing acid from all of the following substrates: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D- mannitol, N-acetyl glucosamine, arbutin, esculin, potassium gluconate. Preferably the bacteria derived from L. suantsaii strain UNI 1801 are capable of converting (fermenting) all of the following substrates: L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D- mannitol, N-acetyl glucosamine, arbutin, esculin, potassium gluconate. Preferably acid production is measured by API 50 CH tests. Preferably the fermentation profile of the bacteria derived from L. suantsaii strain UNI 1801 as measured by API 50 CH test is identical with the fermentation profile of the bacteria belonging to L. suantsaii strain UNI 1801. Preferably the API 50 CH test is carried out by using API 50 CH test strips of Biomerieux SKU Number: 50300.

[0017] The bacteria belonging to L. suantsaii strain UNI 1801 or the bacteria derived from L. suantsaii strain UNI 1801 may be used for e.g. producing a pepper condiment with a decreased salt content. The invention further provides a composition comprising bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801.

[0018] The invention further provides the use of bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 for producing a food product. The invention further provides the use of bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived fromL. suantsaii strain UNI 1801 for preserving a food product. The invention further provides the use of bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 for fermenting a food product. The invention further provides the use of bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 for decreasing the pH of a food product.

[0019] During the use conditions under which the bacteria are able to convert a carbohydrate into an acid are used.

[0020] The invention further provides a method for producing a food product, comprising adding bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 to the raw material of the food product. The invention further provides a method for preserving a food product, comprising adding bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 to the raw material of the food product. The invention further provides a method for fermenting raw material of a food product, comprising adding bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 to the raw material of the food product. The invention further provides a method for lowering the pH of a food product, comprising adding bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801 to the food product.

[0021] The method according to the invention is carried out under conditions enabling the bacteria to convert a carbohydrate into an acid.

[0022] The invention further provides a food product comprising bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801.

[0023] The invention also provides a food product, which is produced by the use of bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from L. suantsaii strain UNI 1801.

[0024] The food product preferably comprises plant material, preferably vegetable, highly preferably pepper. The food product preferably consists of plant material, preferably vegetable. The food product preferably consist essentially of ingredients derived from plants (plant material), preferably e.g. consists at least 75% by weight, at least 80% by weight or at least 85% by weight of ingredients of plant origin. Preferably the plant material comprises a vegetable. Preferably the vegetable comprises pepper, preferably ground or chopped pepper. Preferably the plant material comprises a mixture of more than one vegetable types. Preferably the more than one type of vegetables comprises e.g. pepper, onion, garlic. Preferably the food product is pepper (paprika) paste. Preferably the food product consists essentially of pepper, preferably consists of at least 75% by weight, at least 80% by weight or at least 85% by weight of pepper.

[0025] The use of L. suantsaii for fermenting pepper is also provided. The use of L. suantsaii for converting sugars in pepper into the corresponding acids is also provided. The use of L. suantsaii for producing a pepper condiment is also provided. Preferably the L. suantsaii bacteria are bacteria belonging to L. suantsaii strain UNI 1801 or bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801. Preferably the pepper condiment produced by the use of L. suantsaii comprises no more than about 11% (w / w), no more than about 10% (w / w), no more than about 9% (w / w), no more than about 8% (w / w), no more than about 7% (w / w), preferably about 5-11% (w / w), about 5-9% (w / w), about 5-7% (w / w), about 7-11% (w / w), about 9-11% (w / w) of NaCl.

[0026] Preferably the pH of the pepper condiment is at least about pH=3.2, at least about pH=3.4, at least about pH=3.5, at least about pH=3.6, at least about pH=3.7, highly preferably at least about pH=3.8 or highly preferably about pH=3.5-3.8, highly preferably about pH=3.2-4.0, highly preferably about pH=3.6-4.0. Preferably no acidity regulator agent is added to the pepper condiment during its production.

[0027] Preferably the bacteria are lyophilized. BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 Results of the analytical tests carried out during fermentation of the samples. A: LI, B: L2; C: L3; D: L4, x axis: day of sampling, y axis: concentration

[0029] Figure 2 Colony of L. suantsaii UNI 1801 in a MRS 5 dish

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] Bacteria of L. suantsaii strain UNI 1801 are Gram positive, rod shaped bacteria forming creamwhite colonies with two types of morphology (regular round and irregular edges) on MRS 5 medium. L. suantsaii strain UNI 1801 are facultative anaerobic, mesophilic (temperature optimum of 28.5°C), heterofermentative (CO2 gas production can be measured during the lactic acid fermentation of glucose) bacteria. The pH range of 5.8-6.0 is optimal for its growth. Salt tolerance: L. suantsaii strain UNI 1801 can tolerate a maximum NaCl content of about 11% by weight; salt tolerance cannot be increased with glycine -betaine as an osmoprotective additive. L. suantsaii strain UNI 1801 may be used for the production of food products, such as condiments produced from pepper or other vegetables. The terms pepper condiment and pepper paste are used interchangeably herein, unless otherwise indicated, pepper condiment typically contains ground or chopped paprika. Pepper condiment typically contains table salt. The pepper condiment preferably consists essentially of pepper (paprika) or pepper (paprika) and table salt or pepper (paprika), table salt and water. The pepper is preferably ground or chopped. Pepper condiment may be produced, for example, in the following way:

[0032] - mixing pepper with table salt (NaCl), thus producing a mixture,

[0033] - grinding the mixture.

[0034] According to another embodiment, the pepper or other vegetables are first ground and then mixed with salt.

[0035] The pepper may be raw or heat-treated, preferably raw. In addition to salt, other additives may be added to the pepper, such as acidity regulator agents used in food production (e.g. citric acid), preservatives used in food production (e.g. potassium sorbate), emulsifiers or thickening agents used in food production (e.g. xanthan gum). The pepper condiment may contain other vegetables besides paprika. The other vegetables are preferably added to the pepper before the salt treatment.

[0036] The mixture can be heat-treated if necessary.

[0037] L. suantsaii UNI 1801 can be added to pepper (or a mixture of pepper and other vegetables) before mixing with salt or to the mixture. Preferably, L. suantsaii UNI 1801 is added to the mixture, thereby producing an inoculated mixture. The inoculated mixture is preferably stored under conditions under which L. suantsaii UNI 1801 is able to convert the carbohydrate in the inoculated mixture into acid.

[0038] Water may be added to the pepper, mixture or inoculated mixture. Water may be added at any time during the preparation of the pepper condiment.

[0039] For the production of traditional Hungarian pepper condiments, at least 10% by weight of NaCl must be added to avoid product deterioration caused by microorganisms. Typically, however, the proportion of NaCl is even higher, reaching up to 25% by weight.

[0040] When L. suantsaii UNI 1801 is used for the production of the pepper condiment, the amount of NaCl is added in such a way that its amount is no more than 11% by weight, preferably no more than 10% by weight, preferably no more than 9% by weight, preferably no more than 8% by weight, especially preferably no more than 7% by weight, particularly preferably 5-7% by weight.

[0041] When an acidity regulator agent is also added to the pepper or the mixture during the production of the pepper condiment, the amount of the acidity regulator agent is preferably adjusted so that the pH is such that it inhibits the growth of pathogenic microbes, but allows the growth of the L. suantsaii bacterium. Preferably, the pH of the food product or food product raw material when the L. suantsai bacterium is added is at least pH=3.8, at least pH=3.9, at least pH=4 or between pH=3.8 and pH=5 or pH=3, between 8 and pH=4.8 or between pH=4 and pH=4.5.

[0042] The inoculated mixture is preferably stored under conditions under which L. suantsaii UNI 1801 bacteria are able to ferment the carbohydrate content of the mixture. Surprisingly, we have found that fermentation in an inoculated pepper mixture stops without intervention at a pH value of about 3.6-4.0, so the pepper condiment fermented with L. suantsaii UNI 1801 is not more sour than the the pepper condiment produced by the traditional method, without the use of L. suantsaii UNI 1801.

[0043] NaCl tolerance (salt tolerance, weight / volume % concentration): the highest NaCl concentration at which the bacterium is still able to grow.

[0044] Bacteria from the L. suantsaii strain UNI 1801 are, for example, bacteria that were obtained by culturing or modifying (e.g. by genetic modification or selection) bacteria belonging to L. suantsaii strain UNI 1801.

[0045] EXAMPLES

[0046] L. suantsaii UNI 1801 bacteria were inoculated into ground pepper. The samples are summarized in Table 1: Table 1

[0047] The heat treatment was carried out in the usual way during the industrial production of pepper paste: the sample was heated to 90 °C, followed by a 20-minute hold.

[0048] Table 2. UNI 1801 cell numbers inoculated into the ground pepper

[0049] The samples were stored at room temperature.

[0050] Table 3 pH values measured in the samples during fermentation

[0051] Analytical tests

[0052] In the analytical tests, we have seen that fermentation started in all four systems (L1-L4), and differences between the time courses were seen (approx. 2 week shift), which can be explained by the different salt content of the samples (for the systems with higher salt content, L3 and L4). (Figure 1)

[0053] Test conditions for organic acid tests: HPLC device: Agilent Technologies 1260 Infinity degasser, binary pump, autosampler, column thermostat, detectors.

[0054] The automatic sample dispenser injected 50 pl of the sample. The measurement was performed isocratically, the eluent was 0.01 N sulfuric acid. The flow rate was 0.6 ml / min. The separation was carried out by a Phenome-nex Rezex Monosaccharide H+ (300x7.8mm) column, the column temperature was 50°C. Detection was performed with a DAD detector at 205 nm. The duration of the analysis was 30 minutes.

[0055] The measurements were performed with a 6-point, external standard calibration, calibration range 5-1500 mg / 1. Tested components: oxalic acid, citric acid, pyruvic acid, lactic acid, formic acid, acetic acid, propionic acid, isobutyric acid and butyric acid. The accuracy of the measurement is ±10%.

[0056] Test conditions for sugar measurement:

[0057] An Agilent Technologies 1260 Infinity high-performance liquid chromatograph connected to an Agilent UV and RID detector was used for the measurements.

[0058] The automated sample dispenser (Agilent Technologies 1260 ALS) injected 50 pl of the samples. The measurement was performed isocratically, the eluent was HPLC-grade water, and the flow rate was 0.6 ml / min. The separation was completed by a Rezex RPM-Monosaccharide Pb+2 (300 x 7.8 mm) column, the column temperature was 80 °C. Detection was performed with a RID detector, the temperature of which was 40 °C. The duration of the analysis was 40 minutes.

[0059] The concentrations of the following compounds were examined: sucrose, maltose, lactose, glucose, galactose, fructose and mannitol. The measuring range of the method was 5-2000 mg / 1, except for fructose (15-2000 mg / 1). The accuracy of the measurement was ±10%.

[0060] Sensory examination

[0061] Gas production was observed in samples LI and L2 on day 28 before sampling. Opening the bottles was accompanied by a loud pop. In the case of samples L3 and L4, gas production was observed only when sampling on day 42, which supports the slower fermentation.

[0062] Isolation of L. suantsaii UNI 1801 from the samples

[0063] 1 g of the ground pepper sample was taken up in 9 ml of peptone water, then 100 pl of the suspension was spread on MRS5 dishes (on days 13, 21, 28, 36, 42). The dishes were incubated at 28°C for 3 days.

[0064] Table 4. Number of lactic acid producers lactic acid bacteria pcs / g

[0065] The colony morphology characteristic of strain UNI 1801 (Figure 2) was recognized in the MRS5 dishes (on day 21), colony PCR was made from the colonies (sampling was done from all dishes), and Real-Time PCR (qPCR)was performed with primers specific for Lactobacillus suantsaii UNI 1801. Based on the analysis, we can say with certainty that the lactic acid bacteria that grew in the peppers are of the UNI 1801 strain.

[0066] On the 36th and 42nd days, colony morphologies different from Lactobacillus suantsaii strain UNI 1801 were identified in the dishes. We performed Real-Time PCR (qPCR) with specific primers for the lactic acid bacterium Lactobacillus suantsaii UNI 1801, isolated DNA from the colonies, and then amplified it with specific primers for the 16S rDNA (1500bp fragment) regions. The amplicons were then sequenced and the resulting sequence was analyzed with the NCBI Nucleotide Blast database. The nucleotide composition of the 16S rDNA region of the isolated bacteria and the qPCR analysis confirmed that the strains isolated from the peppers, despite the different colony morphology, are identical with the UNI 1801 strain.

[0067] API 50 CH: az izolalt tejsavbakteriumok szenhidrat metabolizmusdnak vizsgdlata

[0068] API 50 CH: analysis of carbohydrate metabolism of isolated lactic acid bacteria

[0069] Rapid identification assays used in microbiology allow the identification / comparison of bacteria in specific taxonomic groups within 24-48 hours (in our case 72 hours) by examining pure cultures of bacteria. Prefabricated test strips were used for this purpose. The plastic strips supplied by the manufacturer (bioMerieux) contain the substrates needed to test the enzyme activity and sugar-metabolizing capacity of the microbes in dry form (lyophilised). During the tests, the test strips are inoculated with a suspension of pure bacterial cultures, which also ensures rehydration of the substrates. Acid production from the substrates on API 50CH strips and growth of the strains could be observed. After inoculation and thermostating of the multitests, the read results were summarized in Table 5.

[0070] Table 5 Acid production of L. suantsaii UNI 1801 from substrates after 72 hours of incubation at 28 °C

[0071]

[0072] Production of pepper condiment

[0073] - Washing raw peppers.

[0074] - Adding about 5-9% by weight of NaCl to the raw pepper, optionally adding other additives (e.g. an acidity regulator agent, a preservative, an emulsifier or a thickening agent) and mixing.

[0075] - Grinding the mixture to the desired particle size.

[0076] - Adding L. suantsaii UNI 1801 inoculum to the ground mixture.

[0077] - Filling the ground mixture into a container under conditions that allow L. suantsaii UNI 1801 to ferment the carbohydrates in the mixture.

[0078] In the container, the bacterial strain UNI 1801 partially ferments the sugar content of the pepper and, unexpectedly, around pH=3.6-4.0, the process stops without intervention, so the flavoring function of the pepper product will not be inferior to that of the conventional product due to it being more acidic.

[0079] The carbon dioxide produced by the bacterial strain displaces the air (oxygen) dissolved in or mixed into the ground pepper during the mixing and pumping process, contributing to improving the quality of the product and its stability against oxidative deterioration. To this end, the container must ensure the free removal of the carbon dioxide gas produced. The progress of the partial fermentation process, its completion or cessation, can be easily monitored by measuring the pH. The time required depends on the parameters of the raw material and the recipe used (salt, acid addition) and is approximately 1-2 months.

Claims

CLAIMS1. Use of Lactobacillus suantsaii for producing a fermented, pepper based food product.

2. Bacteria belonging to Lactobacillus suantsaii strain UNI 1801 having the accession number of NCAIM (P) B 001503.

3. Bacteria derived from the bacteria belonging to Lactobacillus suantsaii strain UNI 1801 according to claim 2, wherein the bacteria derived from the bacteria belonging to L. suantsaii strain UNI 1801 have a NaCl-tolerance of at least 5% (w / w), preferably at least 7% (w / w).

4. Bacteria derived from the bacteria belonging to Lactobacillus suantsaii strain UNI 1801 according to claim 2 or the bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801 according to claim 3, wherein the bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801 are capable of converting one or more of the following substrates: citric acid, L-arabinose, ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannitol, N-acetyl glucosamine, arbutin, esculin, potassium gluconate.

5. Bacteria derived from the bacteria belonging to Lactobacillus suantsaii strain UNI 1801 according to claim 2 or the bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801 according to claim 3 or 4, wherein the fermentation profile of the bacteria derived from bacteria belonging to L. suantsaii strain UNI 1801 is identical with the fermentation profile of bacteria belonging to L. suantsaii strain UNI 1801, wherein the fermentation profile is measured by API 50CH test.

6. Use of the bacteria according to any one of claims 2-5 for the production, fermentation, conservation or decreasing the pH of a food product.

7. The use according to claim 6, wherein the food product comprises vegetable, preferably pepper Capsicum annuuni).

8. The use according to claim 6 or 7, wherein the food product consist of at least 75% by weight of plant material.

9. Food product, which is produced by the use of the bacteria according to any one of claims 2-5.

10. A composition comprising the bacteria according to any one of claims 2-5.

11. The use according to any one of claims 6-8, the food product according to claim 9 or the composition according to claim 10, wherein the pH value of the food product or the composition is at least 3.4, preferably at least 3.6.

12. The use according to any one of claims 6-8 and 11, the food product according to claim 9 or 11 or the composition according to claim 10 or 11, wherein the salt content of the food product or the composition is not more than 11% (w / w), preferably not more than 10% (w / w).

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