Valuing vegetable sidestreams

Abstract

The present invention relates to a method for valorizing vegetable sidestreams, the method comprising: (a) mixing vegetables with a composition comprising Lactobacillus reuteri; and (b) fermenting the mixture, thereby obtaining a vegetable paste. Fermentation takes only 24-44 hours and does not require the addition of water, heat or nutrients.

Description

[Technical field]

[0001] The present invention relates to a method for valorizing the food sidestream and to valuable products produced in the food sidestream, in particular the valorization of the vegetable sidestream using Lactobacillus reuteri to improve the nutritional value, flavor or shelf life of foods. [Background technology]

[0002] Food waste, which refers to non-consumable food such as surplus fruits and vegetables that are dumped or discarded by farmers or retailers, bruised fruits and vegetables, or food that is discarded after purchase, is becoming a serious problem that is increasingly unacceptable. With world hunger still increasing (Food and Agriculture Organization of the United Nations, 2011 report), about one-third of the food produced in the world is thrown away. Food waste is also highly perishable, attracting animals, and leading to odor pollution.

[0003] Instead of being thrown away, food has been used as animal feed. CN104982658 describes fruit and vegetable waste that is turned into biological feed by pasteurization at 80-100°C for 3-6 hours, followed by fermentation with a bacterial mixture for 50-70 hours. In this way, valuable compounds present in food waste are used for animals.

[0004] Another way to extract value from food waste is to subject it to anaerobic digestion in fermenters. EP1149805 describes the anaerobic digestion of dewatered vegetable waste. Anaerobic digestion produces biogas and fertilizer.

[0005] Converting food into animal feed, biogas or fertilizer extracts more value from food than if it were simply wasted, but food is still lost from the food chain for human consumption. It would be preferable to keep food in the food chain and reuse it for human consumption. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention The present invention provides a method for fermenting vegetables, comprising the steps of: (a) Supplying vegetables; (b) mixing the vegetables of (a) with a composition comprising Lactobacillus reuteri; (c) fermenting the mixture obtained in (b) for 24 to 44 hours, thereby obtaining a vegetable paste; wherein the mixture is fermented without the addition of water, nutrients, heat or oxygen.

[0007] The method has many advantages. First, it allows a high-value use of food that would otherwise be wasted and unavailable for human consumption, i.e. valorization of vegetable sidestreams. The method can be carried out at ambient temperature, i.e. without heating, aeration and prior sterilization or pasteurization of the vegetable sidestream. Yet another advantage is that the method requires a relatively small investment, since it does not require expensive or sophisticated equipment and can be used at the same location where the waste is generated. This also means that transportation may be reduced and contributes less to the carbon footprint compared to current methods that require the transport of waste to a dedicated location. As there is no need to add additional water, thermal energy (heat) to the fermenter, the method according to the invention saves a lot of time, money and energy. The invention also creates a more attractive market and added value for primary producers, and at the same time allows functional improvement of various food products.

[0008] The products obtained by the method according to the invention may be used in food products, including food ingredients, to improve their flavor, nutritional value and long-term shelf life. As is well known to those skilled in the art, the products obtained by the method according to the invention may also be used to improve feed products, including feed ingredients. The improvement is always relative to a similar food or feed product to which no product according to the invention has been added.

[0009] In the context of the present invention, the term "vegetables" includes crops such as eggplant, bell pepper, broccoli, cauliflower, chives, zucchini, cucumber, endive, garlic, leek, lettuce, corn, onion, potato, pumpkin, tomato, etc. In one embodiment, the vegetables are selected from tomato, bell pepper, broccoli, cabbage. The plant-based material may consist of one vegetable (e.g., 100% w / w tomato, 100% w / w broccoli, 100% w / w bell pepper, or 100% w / w cabbage). In another embodiment, the plant-based material comprises different vegetables, i.e., a mixture of vegetables. For example, 70% w / w bell pepper and 30% w / w broccoli or 30% w / w cabbage, based on the total weight of the vegetables. In another embodiment, the vegetable mixture comprises 10-40% w / w bell pepper, 10-40% w / w broccoli, 10-40% w / w tomato, and 10-40% w / w white cabbage, based on the total weight of the vegetables. In practice, the exact vegetable percentages are often unknown, but the method can still be effectively applied with good results leading to the product of the invention.

[0010] The vegetables are preferably raw, i.e. unheated, unsterilized, unpasteurized, unsalted and unprocessed vegetables. Pre-treated or processed vegetables can be used in the method of the invention without compromising the quality of the final product, but pre-treatment is not necessary and makes the method unnecessarily complicated and expensive. The vegetables are preferably only reduced in size by cutting or blending. The size of the vegetables is preferably 0.1-10 cm square, more preferably 0.1-1.0 cm square, most preferably 0.1-0.2 cm square.

[0011] The vegetables may be stored in open, semi-open, or sealed containers. Light is not required. There is no need to pressurize, add oxygen, or protect the vegetables from oxygen. The vegetables are preferably fermented as quickly as possible to minimize spoilage.

[0012] In the method according to the invention, preferably vegetable waste, i.e. vegetables that cannot be consumed, are used. This includes vegetables discarded by farmers or retailers, blemished vegetables, sometimes referred to as vegetable waste. In this way, vegetable side streams may be valorized using the method according to the invention.

[0013] Preferably, the vegetables are used in the process of the present invention as soon as possible to minimize spoilage, e.g., within 1-7 days, 1-3 days, 48 ​​hours, 30 hours, 24 hours, 18 hours, or 10 hours after storage, rejection, or abandonment.

[0014] The vegetables are used as is, cut or blended, without any additions except for a composition comprising or consisting of Lactobacillus reuteri, which can be fully fermented within 44 hours, preferably within 20-40 hours or 20-30 hours, more preferably within 24 hours or 25 hours. The mixing and fermentation are carried out at ambient temperature. No heating or temperature control is required. The ambient temperature without heating will depend on the local climate and season, and is preferably in the range of 18°C ​​to 35°C. In one embodiment, the fermentation temperature is in the range of 20°C to 30°C or 20°C to 25°C. More preferably, the fermentation temperature is 23°C. The fermentation is carried out at ambient oxygen levels. No exclusion or addition of oxygen is required.

[0015] During fermentation, the pH is not controlled and will decrease as a result of acid formation, particularly lactic or acetic acid. These acids are formed during fermentation and remain in the fermentation end product. The pH is between 3 and 4, such as 3.5 and 4.0, at the end of the fermentation. In one embodiment, the pH decreases to pH 3.8. Typically, the pH minimum is reached within 44 hours, preferably within 20 to 40 hours or within 20 to 30 hours, more preferably within 24 hours or within 25 hours. Fermentation according to the process of the invention is evident from an increase in Lactobacillus reuteri CFU and an increase in lactic acid, acetic acid and reuterin levels, preferably within 24 hours from the start. In one embodiment, the Lactobacillus reuteri concentration is 10E8-10E9 CFU / ml within 24 hours. At the end of the fermentation, the lactic acid level is 2-20 gr / l, the acetic acid level is 1-5 gr / l and the reuterin level is preferably 1-50 mM. The vitamin B12 level after fermentation is preferably 1-8 μg per paste, for example 2-5 μg per paste. Preferably, no propionic acid is formed and the propionic acid level is less than 0.2 g / l. The metabolite levels, such as glucose, fructose, lactic acid, acetic acid, propionic acid, mannitol or reuterin levels, may be determined by any convenient method. In one embodiment, the metabolite levels are determined by HPLC. The CFU are preferably determined by bacterial plate count.

[0016] The vegetables are preferably mixed with a composition comprising or consisting of Lactobacillus reuteri. No other bacteria need to be added. Any strain of Lactobacillus reuteri may be used. Lactobacillus reuteri is capable of producing the broad-spectrum antibiotic reuterin (3-hydroxypropionaldehyde) when fermented on glycerol and may be isolated from a suitable source such as the human or animal digestive tract or may be obtained from an international collection of microbial strains, such as DSMZ (DSMZ, Braunschweig, Germany) or ATCC (ATCC, Manassas, Virginia, USA). A strain of Lactobacillus reuteri capable of producing vitamin B12 is preferred. Such a strain of Lactobacillus reuteri contains a functionally active vitamin B12 biosynthetic gene cluster encoding all the enzymes required for the synthesis of vitamin B12. Strains of Lactobacillus reuteri capable of producing vitamin B12 are known in the art, and examples include Lb.reuteri ATCC 55730, Lb.reuteri ATCC 6475, Lb.reuteri CRL 1098, Lb.reuteri DSM 12246, Lb.reuteri DSM 16143, Lb.reuteri DSM 17938, Lb.reuteri DSM 20016 / JCM1112, Lb.reuteri DSM 23877, Lb.reuteri DSM 23878, Lb.reuteri DSM 23879, Lb.reuteri DSM 23880, and the like.

[0017] Vitamin B12 or cobalamin refers to any form of vitamin B12, such as cyanocobalamin, hydroxocobalamin, methylcobalamin, or 5'-deoxyadenosylcobalamin, depending on the group that binds to cobalt. Assays for determining cobalamin concentration are known in the art, such as bioassays, HPLC, or LC / MS. An example of a suitable bioassay is the growth of a microorganism that requires cobalamin for growth in a vitamin B12-free medium, such as the L. delbrueckii assay. When using LC / MS, the Waters® Atlantis TM The C18 column was a Waters Micromass® ZQ TM Detection is preferably performed in conjunction with a 4000 single quadrupole mass spectrometer, with analysis conditions being acetonitrile / water in binary gradient mode, without the need for buffers or ion-pairing reagents (Waters, Milford, USA).

[0018] The composition may consist of or comprise Lactobacillus reuteri. In one embodiment, the composition comprises at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w or at least 95% w / w of bacteria, such as 90% w / w to 99.8% w / w or 95% w / w to 99% w / w, by dry weight of the composition. All of the bacteria in the composition are Lactobacillus reuteri. In one embodiment, a single strain of Lactobacillus reuteri is used. In another embodiment, a mixture of Lactobacillus reuteri strains is used.

[0019] In the context of the present invention, dry weight or dry matter may be determined by methods known in the art, typically involving removing all or at least 98%, at least 99% of the water in the sample, for example by drying a representative sample in an oven until the weight is constant and comparing the weight of the sample before and after drying. Drying may take from a few minutes to a few hours, for example 10 minutes to 24 hours, depending on the drying temperature, water content and sample size. In one embodiment, dry weight is determined by drying a 1-100 ml sample in a conventional oven at 90-105° C. for about 2-24 hours until the weight is constant. Determining wet weight does not require drying, and usually only requires separation of the broth.

[0020] In one embodiment, the composition added to the vegetables is a wet biomass (or pellets) of a Lactobacillus reuteri monoculture fermentation. In one embodiment, the wet biomass is a fermented biomass washed with saline before being added to the vegetables. Preferably, the wet biomass is added to the raw vegetables at a concentration of 0.1% w / w to 0.5% w / w, e.g. 1% w / w to 5% w / w or 3% w / w to 5% w / w, based on the weight of the raw vegetable. No additional water needs to be added.

[0021] Lactobacillus reuteri has the advantage of producing a variety of vitamins such as folic acid, biotin, riboflavin, and vitamin B12, as well as antibacterial compounds such as lactic acid and acetic acid. Another advantage is that scale-up is relatively easy and can be reliably achieved with lactic acid bacteria, in part because these cultures do not require aeration.

[0022] A vegetable paste obtained by the method according to the invention is another aspect of the invention. The vegetable paste has a very good fermented flavour. This is achieved without adding any additional water, nutrients, herbs, spices or flavourings or other microorganisms apart from Lactobacillus reuteri. The vegetable paste comprises cobalamin, reuterin, lactic acid and acetic acid, which are responsible for the nutritional value and antibacterial activity of the vegetable paste end product. In one embodiment, the vegetable paste has a dry matter content of at least 50% w / w, preferably the vegetable paste has a dry matter content of at least 55% w / w, at least 60% w / w, at least 65%, at least 70% w / w or at least 75% w / w, up to 80% w / w, for example a dry matter content of 55% w / w to 80% w / w, 55% w / w to 70% w / w or 55% w / w to 65% w / w. The dry matter content of the paste may be determined by methods known in the art, as described above.

[0023] The vegetable paste may be used as such or formed into a liquid or powder. Preferably, the vegetable paste may be dried to kill any microorganisms. Spray drying or freeze drying are preferred drying techniques.

[0024] The resulting paste, powder or liquid may be used in food or feed applications, for example as a food or feed product, including a food or feed ingredient, preferably a vegetable food product.

[0025] The use of the vegetable paste according to the invention, after suitably forming into a powder or liquid, is of great advantage for the following reasons: the vegetable paste may be used as a food ingredient to add or improve the flavour, to enhance the nutritional value, to increase the vitamin content or to increase the shelf life of existing and new food products, or as a meat substitute, since the vegetable paste has a very good fermented flavour and is rich in vitamin B12. The vegetable paste, after suitably fully or partially drying, may be added to food products, such as soups, sauces and spreads, especially foods containing vegetable blends. The appropriate concentration depends on the food or feed application. In one embodiment, the concentration varies between 1 and 10% w / w, based on the dry weight of the food or feed. In one embodiment, the paste, although in vegetable form, is used as a flavouring, similar to a stock, and is used in soups, sauces or stews. Of course, bones may be added to the paste if desired. In another embodiment, the vegetable paste is used to increase the vitamin B12 content of meat substitutes, such as vegetable or vegetarian products. In another embodiment, the vegetable paste is used in or as a meat substitute. In another embodiment, the vegetable paste is used to enhance the flavor of food for people who have lost their sense of taste or smell, such as those undergoing chemotherapy, the elderly, or those suffering from anosmia or hyposmia. In another embodiment, the paste is used to simultaneously enhance the flavor, vitamin content, and shelf life of a food. These modified food or feed products, modified by the addition of the powder, paste, or liquid according to the present invention, and having enhanced vitamin B12 content, flavor, or shelf life, are also an aspect of the present invention. EXAMPLES

[0026] (Determination of Metabolites) Metabolite levels such as glucose, fructose, lactate, acetate, propionate, mannitol or reuterin are determined by HPLC. Vitamin B12 analysis (Table 6) was performed using an LC / MS Waters® Atlantis TMA C18 column was used for detection using a Waters Micromass® ZQ TM The analysis was performed on a 4000 single quadrupole mass spectrometer (Waters, Milford, USA).

[0027] Example 1: Laboratory-scale fermentation of Lactobacillus reuteri Approximately 30 g of unheated blended (Thermomix TM5, Cnudde BV, The Netherlands) tomatoes were fermented with 1.5 ml of unheated grown and washed preculture of Lactobacillus reuteri either fresh (F) or pre-pasteurized at 100°C for 10 min (S) at 23°C or 30°C. The results are shown in Table 1 (control, no inoculation), Table 2 (30°C) and Table 3 (23°C) and show that both glucose and fructose were converted simultaneously, fructose was converted slightly faster than glucose, fresh (unheated) tomatoes were fermented faster than pasteurized tomatoes, and fermentation with Lactobacillus reuteri was faster at 30°C than at 23°C. In addition to lactic and acetic acid, mannitol was also the main fermentation product with production levels up to 14gr / l.

[0028] F: raw, vegetables are used "as is", i.e. without heat treatment, without adding additional water or nutrients, just cut or blended; S: pasteurized at 100°C for 10 minutes; nd not detected. [Table 1] [Table 2] [Table 3]

[0029] Vitamins normally produced after fermentation with Lactobacillus reuteri remained, especially folic acid, biotin, riboflavin and vitamin B12. The vitamin B12 content in tomato paste after fermentation with Lactobacillus reuteri was 3 μg / kg paste. Similar results were obtained in the fermentation of bell peppers and white cabbage.

[0030] In conclusion, good precision fermentation can be performed on vegetables using Lactobacillus reuteri (for vitamin B12 production, antibacterial and reuterin production) even at room temperature without heat treatment and without adding additional water to the vegetables.

[0031] Example 2: Fermentation of mixed vegetables This experiment demonstrates that the method of the present invention also works on mixed vegetables. Raw vegetables (white cabbage, tomato, capsicum, and broccoli) were blended in a 1:1:1:1:1 ratio by weight using a domestic blender. Immediately after blending, 50 g portions of the blended vegetable mixture were dispensed into sterile plastic cups and stored at -40°C until the start of the fermentation experiments. Lactobacillus reuteri DSM strain 122.46 was cultured overnight on standard MRS medium (Sigma-Aldrich, Steinheim, Germany). Cells from mature cultures were harvested by centrifugation, washed, and resuspended in an equal volume of 50 mM potassium phosphate buffer, pH 6.8. For fermentation, the raw, i.e., unheated, blended vegetable mixture was inoculated with washed and overnight cultured Lactobacillus reuteri (5% w / w, wet weight), in duplicate, and subsequently incubated at room temperature (22°C) and 30°C. To examine the need for additional glucose addition, a vegetable mix containing an additional 2% w / w (wet weight) glucose was also incubated at both temperatures. As a control, a raw, unpasteurized vegetable mix was also incubated at the two temperatures without inoculation with Lactobacillus reuteri.

[0032] The experiment was performed twice and the average values ​​are shown in Table 4. The decrease in pH is an indicator of acid product formation, i.e., fermentation. Despite the absence of added sugar, the blended vegetable mixture was almost completely fermented (pH decreased from 4.8 to below 4.0) within 24 hours of incubation. In the state of the art, nutrients are typically added to promote fermentation. However, the results show that not adding glucose had no significant effect on acidification or cell growth, which is very practical and cost-effective. [Table 4] [Table 5]

[0033] The results also show that no heat is required for fermentation when using the method of the present invention: the blended vegetable mixture was almost completely fermented (pH decrease) both at room temperature and at 30°C.

[0034] The observed fermentation was clearly caused by the addition of Lactobacillus reuteri preculture, as the control showed almost no acidification. The pH did not decrease significantly and remained above pH 4.5, regardless of whether additional glucose was added or not. Table 5 shows the acid formation and bacterial growth during fermentation. In the control (no inoculation), no lactic or acetic acid production was evident and bacterial growth was very limited.

[0035] Regarding sugar consumption and metabolite formation, the amount of glucose consumed during 24 hours of fermentation was about 12-13 g / L with and without the addition of additional glucose, resulting in the production of about 6 g / L lactic acid and 3 g / L acetic acid. Since the fermentation at 30°C was slightly faster and slightly more extensive than that at room temperature, the glucose utilization was also slightly higher, and the productivity of lactic acid and acetic acid was also slightly higher.

[0036] Lactobacillus reuteri cell growth occurred 24 hours after initiation and continued until there were approximately 10E9 CFU (colony forming units) per ml of fermented vegetable. Longer incubation did not result in further growth of the culture, and a clear decrease in cell count was observed, especially at room temperature and in the absence of additional glucose. Vitamin B12 was produced at 5 μg / L by 10E9 Lactobacillus reuteri per ml.

[0037] The results demonstrate that raw vegetables and vegetable side streams can be easily fermented with Lactobacillus reuteri without the need for temperature control or heat, additional water or added sugar. The ferments contained the same amount of metabolites (lactic and acetic acid) and the same number of cells at both incubation temperatures with and without added sugar. Using the method of the present invention, the fermented vegetables had a very good fermented flavor and were rich in vitamin B12 as a result of the survival and growth of Lactobacillus reuteri. No other bacteria, heat, oxygen or nutrients need to be added.

[0038] Example 3: Vegetable blend fermentation on a pilot scale For pilot-scale fermentation, a preculture of Lactobacillus reuteri (DSM 1224, DSMZ, Braunschweig, Germany) was grown on MRS medium (Sigma-Aldrich, Steinheim, Germany) at 37°C until it reached OD600 = 2.0 and used to inoculate a 2-liter overnight culture. The next day, the microbial biomass of the 2-liter culture was harvested by centrifugation (10,000 g for 10 min) and washed with sterile saline (0.9% NaCl).

[0039] Approximately 150 kg of raw vegetable mixture (tomatoes, bell peppers, broccoli, white cabbage) was cut into approximately 1-2 mm cubes using a Finis Cutter pinned grater (spijkerrasp, FINIS, Ulft, The Netherlands), ground and formed into a vegetable puree containing 5.1% dry matter. The puree was added to 100 liters of sterilized fermentation broth without heat treatment and without additional water supply. Two liters of overnight cultured Lactobacillus reuteri biomass were added. No other nutrients were added. The mixture of vegetable puree and microbial biomass was incubated at 30°C under stirring (400 rpm). The pH value at the start was approximately 5.8. During the fermentation, pH, stirring speed and temperature were automatically monitored. Within 24 hours, the pH of the broth reached a minimum value (pH 3.8), indicating that the fermentation was completed within 24 hours. After 44 hours, the fermentation was stopped. The acidic pH at the end of fermentation was mainly due to the formation of lactic and acetic acids. The resulting vegetable paste was analyzed.

[0040] Metabolite analysis confirmed that after one day, the majority of the fermentation was taking place using glucose but not fructose. Lactic and acetic acids were formed. No propionic acid was detected. [Table 6]

[0041] Example 5: Powder according to the invention The vegetable paste obtained after fermentation is converted into a powder by heat evaporation. The powder contains, on a dry weight basis, 15-30 μg of vitamin B12 per kg of powder, 50-100 gr of lactic acid per kg, 20-40 gr of acetic acid per kg, and less than 1 gr of propionic acid per kg. The powder can be suitably added to food or feed to enhance long-term shelf life, nutritional value, especially vitamin B12 content, or flavor.

Claims

1. 1. A method for fermenting vegetables, comprising: (a) Providing vegetables; (b) mixing the vegetables of (a) with a composition comprising Lactobacillus reuteri; (c) fermenting the mixture obtained in (b) for 24 to 44 hours, thereby obtaining a vegetable paste; wherein said mixture is fermented without the addition of water, nutrients, heat or oxygen.

2. The method of claim 1 wherein the vegetables are raw vegetables.

3. 10. The method of claim 1, wherein the vegetables are discarded vegetables.

4. 2. The method of claim 1, wherein the temperature during fermentation is in the range of 20°C to 30°C.

5. 10. The method of claim 1, wherein the vegetable is tomato, bell pepper, broccoli, or white cabbage, or a mixture thereof.

6. 10. The method of claim 1, wherein the vegetable paste contains 1 to 8 μg of vitamin B12 per liter of paste.

7. 10. The method of claim 1, further comprising forming the vegetable paste into a powder or liquid form.

8. A vegetable paste obtainable according to claim 1.

9. 9. The vegetable paste of claim 8, comprising 1-8 μg / L of vitamin B12, 2-20 g / L of lactic acid, 1-5 g / L of acetic acid, and less than 0.2 g / L of propionic acid.

10. 9. The vegetable paste of claim 8, comprising 1 mM to 50 mM reuterin.

11. A powder or liquid obtained by molding the vegetable paste according to claim 8.

12. Use of a vegetable paste, powder or liquid according to any one of claims 9 to 11 in a food or feed product.

13. 13. The use according to claim 12, wherein the food product is a vegetable product.

14. 13. The use according to claim 12 in a soup, sauce, stew or spread.

15. Use of a vegetable paste, powder or liquid according to any one of claims 9 to 11 in a method for improving the flavour, nutritional value or shelf life of a food or feed product.