Continuous fermentation process of meat curing preparations

Abstract

In some aspects, the present disclosure provides advantageous methods for preparing meat curing agents. In some embodiments, a process for continuously converting plant-based nitrates to nitrites comprises: inoculating a source material liquid containing nitrates with a bacterial culture capable of converting nitrates to nitrites; harvesting a first harvest volume of the source material liquid at a harvest rate when the nitrite in the source material liquid reaches a desired concentration; and replenishing the harvest volume of the source material liquid with additional source material liquid containing nitrates at a feed rate, wherein the harvest rate is higher than the feed rate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 038,935, filed June 15, 2020, and U.S. Provisional Application No. 17 / 348,606, filed June 15, 2021, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to compositions for curing meat and livestock products, and methods of making such compositions. Background Art

[0004] Curing is a key preservation technology for meat and livestock products, extending shelf life, enhancing flavor, and stabilizing color. All-natural meat and livestock products are cured using fermented plant-based curing agents containing nitrites. Batch fermentation is a common process for producing plant-based fermented curing agents. As demand for all-natural meat and livestock products grows, these processes are unable to keep up with the demand for curing agents. Therefore, improved methods for producing plant-based curing agents are needed. Summary of the Invention

[0005] In some aspects, the present disclosure provides advantageous methods for preparing meat curing agents. In some embodiments, such a process for continuously converting plant-based nitrates to nitrites comprises: inoculating a source material liquid with a bacterial culture, wherein the source material liquid contains a concentration of nitrates and the microbial culture is capable of converting nitrates in the source material liquid to nitrites; allowing the microbial culture to reach a desired conversion rate to ferment the source material liquid, thereby converting nitrates to nitrites; harvesting a volume of the fermented source material liquid at a first rate; and replacing the volume of the fermented source material liquid with fresh source material liquid at a second rate, wherein the second rate is slower than the first rate.

[0006] In some aspects, the present disclosure provides advantageous methods for preparing meat curing agents. In some embodiments, a process for continuously converting plant-based nitrates to nitrites comprises: inoculating a source material liquid containing nitrates with a bacterial culture capable of converting nitrates to nitrites; harvesting a first harvest volume of the source material liquid at a harvest rate when the nitrite in the source material liquid reaches a desired concentration; and replenishing the harvest volume of the source material liquid with additional source material liquid containing nitrates at a feed rate, wherein the harvest rate is higher than the feed rate.

[0007] In some embodiments, the process further comprises: determining the conversion rate of nitrate to nitrite; calculating the conversion time (Tconversion) required for converting nitrate to nitrite based on the initial concentration of nitrate in the source material liquid and the conversion rate; calculating the harvest volume based on the total volume of the source material liquid and Tconversion; and calculating the feed rate based on the harvest volume and the harvest time.

[0008] In some embodiments, such processes further comprise harvesting one or more additional harvest volumes of source fluid having a desired nitrite concentration at regular time intervals equal to the harvest time.

[0009] In some embodiments, if the nitrate concentration in the harvest volume is lower than the desired nitrite concentration, the process further comprises reducing the feed rate, reducing the harvest volume, or increasing the harvest time. In some embodiments, the nitrate in the source material liquid is a plant-based nitrate. In some embodiments, the process further comprises adjusting the initial concentration of nitrate in the source material liquid. In some embodiments, the conversion rate of nitrate to nitrate is the maximum conversion rate of the bacterial culture for converting nitrate to nitrite. In some embodiments, the process further comprises adjusting one or more of the harvest volume, harvest rate, and feed rate to maintain the maximum conversion rate of nitrate to nitrite. In some embodiments, the process further comprises determining the conversion rate of nitrate to nitrite, and allowing the conversion rate to become the maximum conversion rate of the bacterial culture for converting nitrate to nitrite and then harvesting the first harvest volume.

[0010] In some embodiments, the harvest volume of the fermented source material liquid is 20%-80% of the initial volume of the source material liquid. In some embodiments, the harvest rate is 3-100 times faster than the feed rate. In some embodiments, the nitrate concentration of the source material liquid is at least 100ppm. In some embodiments, the Brix (Degrees Brix) measurement value of the source material liquid is 1-12. In some embodiments, when at least 85% of the nitrate has been converted into nitrite, the required nitrite concentration is reached. In some embodiments, the required harvest time is 30 minutes-2 hours. In some embodiments, the harvest volume of the fermented source material liquid is 40%-60% of the initial volume of the source material liquid. In some embodiments, the harvest rate is 3-100 times faster than the feed rate. In some embodiments, the required conversion rate is the maximum conversion rate. In some embodiments, the volume of the fermented source material is calculated based on the conversion rate and the concentration of nitrate in the source material liquid.

[0011] In some aspects, the present disclosure provides a system comprising: a fermentor configured to hold a source material liquid comprising plant-based nitrates, the fermentor having an inlet configured to provide the source material liquid to the fermentor and an outlet configured to remove the source material liquid from the fermentor; a processor programmed to receive conversion data to monitor the conversion of nitrate to nitrite when the source material liquid is inoculated with a bacterial culture capable of converting nitrate to nitrite; harvesting a first harvest volume of the source material liquid at a harvest rate when the nitrite in the source material liquid reaches a desired concentration; and replenishing the harvest volume of the source material liquid with source material liquid comprising additional nitrate at a feed rate, wherein the harvest rate is greater than the feed rate.

[0012] In some embodiments, the processor is further programmed to determine the conversion rate of nitrate to nitrite; calculate the conversion time (Tconversion) required for nitrate to be converted to nitrite based on the initial concentration of nitrate in the source material liquid and the conversion rate; calculate the harvest volume based on the total volume of the source material liquid and Tconversion; and calculate the feed rate based on the harvest volume and the harvest time.

[0013] In some embodiments, if the nitrate concentration in the harvest volume is lower than the desired nitrite concentration, the processor is further programmed to reduce the feed rate, reduce the harvest volume, or increase the harvest time. In some embodiments, the nitrate in the source material liquid is a plant-based nitrate. In some embodiments, the conversion rate of nitrate to nitrate is the maximum conversion rate of the bacterial culture for converting nitrate to nitrite. In some embodiments, the processor is further programmed to adjust one or more of the harvest volume, harvest rate, and feed rate to maintain the maximum conversion rate of nitrate to nitrite. In some embodiments, the processor is further programmed to determine the conversion rate of nitrate to nitrite and allow the conversion rate to become the maximum conversion rate of the bacterial culture for converting nitrate to nitrite and then harvest the first harvest volume. In some embodiments, the harvest volume of the fermented source material liquid is 20%-80% of the initial volume of the source material liquid. In some embodiments, the harvest rate is 3-100 times faster than the feed rate. In some embodiments, the nitrate concentration of the source material liquid is at least 100 ppm. In some embodiments, the Brix measurement of the source material liquid is 1-12. In some embodiments, the desired nitrite concentration is reached when at least 85% of the nitrate has been converted to nitrite. In some embodiments, the harvest time is between 30 minutes and 2 hours.

[0014] The present disclosure also provides a composition comprising a curing agent, a method for treating meat, livestock, poultry, or seafood products using the instant curing agent, and meat, livestock, poultry, and seafood products treated with the instant curing agent.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the following detailed description, the present disclosure is further described by way of non-limiting examples of exemplary embodiments and with reference to the several drawings indicated, in which like reference numerals represent similar parts throughout the several views, and in which:

[0017] Figure 1 Exemplary systems are provided that can be used to perform the continuous fermentation processes of the present disclosure.

[0018] Figure 2 Exemplary methods for preparing the curing agents of the present disclosure are provided.

[0019] Figure 3 An exemplary flow diagram for performing fermentation steps continuously is provided.

[0020] Figure 4 is a chart showing exemplary feed and harvest rates that result in a volume of fresh feed equal to the volume of harvest.

[0021] Figure 5 A flow chart of an exemplary feedback loop for controlling a continuous fermentation process is provided.

[0022] Figure 6 is a graph showing the continuous conversion of nitrate to nitrite using a starting material containing 40,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure.

[0023] Figure 7 is a graph showing the change in nitrate and nitrite (dry basis) concentration over time in a batch fermentation using a starting material containing approximately 40,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure.

[0024] Figure 8 is a graph showing the continuous conversion of nitrate to nitrite using a starting material containing 70,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure.

[0025] Figure 9 is a graph showing the change in nitrate and nitrite (dry basis) concentration over time in a batch fermentation using a starting material containing approximately 70,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure.

[0026] Figure 10 An exemplary computer system suitable for use with the systems and methods of the present disclosure is shown.

[0027] Although the drawings indicated above illustrate embodiments of the present disclosure, other embodiments are also contemplated as discussed. The present disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other variations and embodiments can be devised by those skilled in the art that fall within the scope and spirit of the principles of the embodiments of the present disclosure. DETAILED DESCRIPTION

[0028] The present disclosure generally relates to a method for producing a food additive for preserving meat and seafood products. The curing agent produced by the method of the present disclosure can include a nitrite content for curing various meats, such as beef, pork, turkey, chicken, products of such meats, and seafood and seafood products. In some embodiments, the curing agent of the present disclosure can be used with organic or all-natural meat, livestock, poultry or seafood. Therefore, it may be desirable that the nitrite in such curing agent be derived from a natural source. In some embodiments, using bacterial cultures or enzymes, nitrate-rich plants can be fermented to convert nitrates into nitrites. The present disclosure provides a continuous fermentation (nitrate to nitrite conversion) process that is more efficient in terms of time and production costs.

[0029] Figure 1 An exemplary system 100 is provided that can be used to perform the continuous fermentation processes of the present disclosure.

[0030] Such a system 100 may include one or more separate operating modules / units: a plant material concentrate preparation module 101, an automatic mixing module 102, a pasteurization module 103, a fermentation module 104, a source material inoculation 104a for the fermentation module 104, an inactivation module 105, a pasteurization module 106, a centrifugation module 107, an evaporation module 108 and a final processing module 109.

[0031] Figure 2 An exemplary method 200 of preparing a curing agent of the present disclosure using the system 100 is provided. The method 200 includes one or more of the following: preparing a source material concentrate comprising natural nitrates 201, adjusting the source material concentrate for solids, nitrates, and pH 202, pasteurizing the source material concentrate 203, fermenting the source material concentrate 204, inoculating the source material concentrate 204a with a culture activated to a logarithmic growth phase, inactivating microorganisms in a removed fermentation broth 205, pasteurizing the fermentation broth 206, centrifuging the fermentation broth 207, evaporating the fermentation broth 208, and processing a curing agent powder or blend 209.

[0032] Step 201 includes preparing a source material containing natural nitrates into a source material concentrate 101. Various nitrate-containing plants can be used as a source of nitrates. For example, the source material can include, but is not limited to, various forms of lettuce, such as romaine lettuce, feta lettuce, iceberg lettuce, as well as cabbage, spinach, celery, beets, beet greens, Swiss chard, cucumbers, eggplants, mushrooms, bell peppers, butternut squash, zucchini, carrots, artichokes, green beans, lima beans, broccoli, cauliflower, kale, mustard, okra, onions, pea pods, black-eyed peas, green peas, potatoes, radishes, sauerkraut, turnips, other edible plants, or combinations thereof.

[0033] The source material concentrate 101 can be prepared in step 201 from whole plants or plant parts (leaves, stems, flowers, seeds), any form of processed plants, such as juice, concentrate, or dry powder, or a combination thereof. When using whole plants or plant parts, the plants can be physically processed, such as cutting, maceration, pressing, solid-liquid extraction, concentration, drying, etc., to prepare the source material concentrate. In some embodiments, the source material concentrate can be prepared from previously processed plants in the form of vegetable juice, vegetable juice concentrate, or powder. In various embodiments, such source materials can have a nitrate concentration of at least 100 ppm, at least 1000 ppm, at least 2000 ppm, at least 5,000 ppm, at least 10,000 ppm, at least 25,000 ppm, at least 50,000 ppm, at least 100,000 ppm, or at least 140,000 ppm. As a non-limiting example, the nitrate concentration in the source material can be in the range of approximately 15,000 ppm and 40,000 ppm.

[0034] In step 202 , which occurs in the automated mixing module 102 , water is added to the source material concentrate 101 to create a source material solution, and the solids, nitrates, and pH of the resulting solution are automatically adjusted.

[0035] The source material liquid contains a high concentration of nitrates. In various embodiments, the source material liquid may have a nitrate concentration of at least 100 ppm, at least 1000 ppm, at least 2000 ppm, at least 5,000 ppm, at least 10,000 ppm, at least 25,000 ppm, at least 50,000 ppm, at least 100,000 ppm, or at least 140,000 ppm. As a non-limiting example, the nitrate concentration in the source material liquid may be in the range of approximately 15,000 ppm and 100,000 ppm. In some embodiments, the nitrate concentration in the source material concentrate 101 may be adjusted to ensure consistent starting material for the process. For example, the concentrate from whole plants may be supplemented with a pre-prepared concentrate or vegetable juice.

[0036] In addition to the concentration of nitrate, the source material liquid can include other soluble solids that are typically measured in Brix using a handheld refractometer. Such solids may include naturally occurring solids in the source material, such as sugars, proteins, and salts. The Brix range can be from 1 to 12, and in some embodiments, from 4 to 7.5. In some embodiments, the Brix range can be between 7 and 7.5. Soluble solids exert osmotic pressure on microorganisms during fermentation. Due to low osmotic pressure, solutions with low soluble solids ferment faster than solutions with high soluble solids. Regulating soluble solids to control the fermentation rate is crucial for controlling continuous fermentation processes.

[0037] In step 202, additional nutrients may be added to the source material solution to supplement the existing macronutrients and micronutrients used for bacterial culture. Examples of macronutrients include common carbon and nitrogen sources. Carbon sources may be monosaccharides or complex sugars, such as glucose, sucrose, and starch. Nitrogen sources may include yeast extract, peptone, and tryptone. Trace elements may include mineral salts, vitamins, and the like.

[0038] Finally, in step 202, the pH of the source material solution is adjusted to ensure appropriate conditions for the bacterial culture to convert nitrates into nitrites. For example, the pH of the source material solution can be slightly acidic (e.g., pH about 5) to slightly alkaline (e.g., pH about 9). In some embodiments, the pH of the source material solution can be substantially neutral, such as between pH 6 and pH 7.

[0039] In step 203, which occurs in the pasteurization module 103, the source material concentrate 101, which has had its pH, nitrate concentration, and Brix adjusted in step 202, is pasteurized to reduce the microbial load in the solution and thereby avoid microbial competition that could adversely affect the nitrate conversion process. The source material liquid can be heated to a temperature and for a time designed to kill bacterial contamination that could adversely affect the nitrate conversion process. In some embodiments, step 203 can be performed in an ultra-high temperature pasteurizer. In some embodiments, the source material liquid can be heated to a temperature greater than about 200°F for 15 seconds, for example, greater than 250°F. ° F and last for 1-2 seconds or longer.

[0040] In step 204, which occurs in the fermentation module 104, the pasteurized liquid source material is fermented and actively growing microorganisms are used to convert nitrates in the liquid source material into nitrites. To this end, the liquid source material in the fermenter is inoculated with a microbial culture from the inoculation module 104a in step 204a to initiate the conversion of nitrates into nitrites.

[0041] Any microorganism capable of converting nitrate to nitrite can be used in the method of the present disclosure. Suitable microorganisms include, but are not limited to, yeast, fungi, and bacteria. The organisms can include, but are not limited to, for example, Escherichia coli, Rhodobacter sphaeroides, Paracoccus pantotrophus, Wautersia eutropha, Bradyrhizobium japonicum, Pseudomonas spp., Campylobacter jejunii, Wollinella succinogenes, Haemophylus influenzae, Shewanella oneidensis, Desulfitobacterium hafniens, Rhodobacter capsulatus, Klebsiella pneumoniae, Bacillus subtilis, Cyanobacteria spp.), Synechococcus spp., genus Haloferax, Haloarcula spp., Thermus thermophilus, Micrococcaceae, including Micrococcus and Staphylococcus, Gram-positive cocci, including Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Staphylococcus, as well as some lactic acid bacteria, M. varians, S. carnosus, S. vitulinus, S. xylosus, or combinations thereof. Also contemplated are embodiments of the disclosure wherein the conversion of nitrate to nitrite is accomplished using a suitable enzyme preparation, typically one comprising a nitrate reductase.

[0042] Microorganisms are maintained in inoculation module 104a until they need to be added to the fermentor tank. The inoculation culture can be activated by hydrating freeze-dried culture or actively growing culture in growth medium. Growth medium can comprise the conventional carbon source and nitrogen source for cultivating microorganisms in the fermentation industry, and is adjusted according to the needs of specific strains. Growth medium can also be customized plant materials with a large amount of carbon source and nitrogen source for optimal growth rate. The culture in inoculation module 104a can be stored at refrigerated temperature until ready to be inoculated in step 204a. In some embodiments, the culture from inoculation module 104a is inoculated in the source material containing nitrate in step 204a, while being in the logarithmic growth phase of bacterial growth to obtain optimal performance.

[0043] In step 204, after step 204a of inoculating the activated microorganisms into a solution containing nitrates, fermentation is allowed to proceed until the desired concentration of nitrates in the source material liquid has been converted to nitrites. In some embodiments, fermentation can be allowed to proceed until all or substantially all of the nitrates have been converted. In some embodiments, fermentation can be allowed to proceed until 85%-95% of the nitrates have been converted. In some embodiments, fermentation can be allowed to proceed until at least 85% or at least 90% or at least 95% of the nitrates have been converted to nitrites. The nitrate concentration in the source material liquid is converted to nitrites at a desired conversion rate. In some embodiments, the desired conversion rate will be the maximum conversion rate for the given conditions. Once the desired concentration of nitrates in the source material liquid has been converted to nitrites, the first harvest can be removed. Thereafter, in some embodiments, harvesting can be performed at regular time intervals.

[0044] In some embodiments, the feed rate is adjusted based on the volume (which is the same as the harvest amount) that must be supplemented over a period of time. Because the microorganisms in the fermentor tank have adapted and are at the highest conversion rate, the additional volume of source material liquid is fermented substantially at the highest nitrate to nitrite conversion rate.

[0045] In step 205, the removed fermentation broth is transferred to the inactivation module 105, where the microorganisms in the fermentation broth are inactivated. Microorganisms can be inactivated by various methods, such as heating, filtration, or pH changes. Inactivation ensures that the microorganisms do not grow and may deplete nitrite. In some embodiments, an alkaline pH of 7.5-12 can be used to inactivate microorganisms.

[0046] The fermentation broth is pasteurized to kill microorganisms in step 206, which occurs in the pasteurization module 106. Pasteurization can be accomplished by an ultra-high temperature pasteurizer or by other pasteurization techniques.

[0047] In step 207, which occurs in the centrifugation module 206, the pasteurized fermentation broth is centrifuged to remove the inactivated microorganisms and any insoluble solids in the fermented liquid. Removal can also be performed by filtration.

[0048] In step 208, which occurs in the fermentation module 108, the fermentation broth undergoes an evaporation process to produce a concentrate of curing agent solids containing nitrite. In some embodiments, the curing agent can remain as a liquid. In some embodiments, the concentration of nitrite is 5,000-100,000 ppm. Other concentration methods, including centrifugal evaporation or reverse osmosis, can also be used to achieve concentration.

[0049] In step 209 that occurs in the final processing module 109, the curing agent solid is further dried and ground to produce a curing agent powder. Drying can be accomplished by vacuum tray drying, vacuum belt drying, freeze drying, spray drying or other drying techniques. In some embodiments, the curing agent powder can then be mixed with other preservatives such as salt. Finally, the product is packaged for storage or transportation.

[0050] Figure 3 An exemplary flow diagram 300 for continuously performing the fermentation step 204 is provided.

[0051] As described above, a volume of source material liquid (Vtotal) 302 is supplied to the fermentor. The source material liquid may have an initial concentration of nitrate (Cnitrate) 301, which will be converted to a desired concentration of nitrite in the fermentor.

[0052] In batch fermentation process, after microorganism is added to the solution containing nitrate, microorganism initially experiences lag phase, i.e., microorganism grows and adapts to the time period of 2-3 hours of fermentation medium (source material liquid). In the logarithmic phase of microbial growth, the conversion rate of nitrate to nitrite increases exponentially with bacterial growth over time. Therefore, depending on the concentration of nitrate in the source material liquid, it usually takes 6-10 hours for nitrate to be fully converted into nitrite, and most of the conversion occurs in the last 1 hour. After nitrate is completed to the conversion of nitrite, the process is stopped by inactivating microorganisms, and the process is repeated for a new batch of microorganisms and source material liquid.

[0053] The methods of the present disclosure advantageously eliminate or at least significantly reduce the initial lag phase and also take advantage of the highest conversion rates after the microorganisms have reached maximum growth during the fermentation process.

[0054] For example, the process begins by inoculating a first batch of source material liquid with a microbial culture, i.e., step 204a. In some embodiments, the inoculation of the microbial culture can occur only in the first batch of source material liquid. The fermentation in step 204 is allowed to proceed until the desired concentration of nitrate in the source material liquid has been converted to nitrite. At this point, unlike a batch process where the fermentation process is completely stopped, in the process of the present disclosure, only a portion of the fermentation liquid is removed from the fermentor and replenished with fresh source material liquid with additional nitrate, thereby allowing the fermentation process to continue in the remaining liquid.

[0055] In step 303, once the source material liquid is inoculated with the bacterial culture and fermentation begins, the nitrate and nitrite concentrations are measured at desired time intervals. In some embodiments, the concentrations may be measured at regular intervals between 10 and 60 minutes. In some embodiments, the concentrations may be measured every 20 minutes. In some embodiments, the nitrate and nitrite concentrations may be measured continuously.

[0056] In step 304, the highest conversion rate of nitrate to nitrite is determined. In some embodiments, the concentrations of nitrite and nitrate detected in step 303 can be plotted against time. The differential increase in conversion rate is measured and the prediction curve is used to estimate the first harvest / transfer time of the fermentor. The measured concentration of nitrite or nitrate in the graph can be compared to the fermentor with the highest R 2 In some embodiments, the maximum conversion rate is realized in the last 1 hour before nitrate is fully converted into nitrite. In some embodiments, the speed that nitrate consumes or the speed that nitrite concentration increases can be used to determine the conversion rate of nitrate to nitrite.

[0057] In step 305, the highest conversion rate is used to determine an estimated time to complete the conversion.

[0058] In some embodiments, such time is calculated using the following formula:

[0059]

[0060] Wherein T conversion (e.g., hours) is the time required for nitrate in the source material liquid to be completely converted into nitrite at a certain conversion rate.

[0061] C Nitrate is the initial concentration of nitrate in the source material solution (ppm), and

[0062] The conversion rate (ppm / hour) is the rate (eg, maximum rate) required for the conversion of nitrate to nitrite by the microbial culture.

[0063] In step 306, the volume of harvest to be removed at regular time intervals can be determined based on the nitrate concentration, the Brix of the feed, and the time to complete conversion. In some embodiments, such volume is calculated using the following formula:

[0064]

[0065] Where Vharvest (gallons / hour) is the volume of harvest per unit time;

[0066] Vtotal (gal) is the total volume of liquid in the fermenter; and

[0067] T conversion (hours) is the time required for nitrate in the source material liquid to be completely converted into nitrite at a certain conversion rate.

[0068] Higher nitrate concentrations may require a smaller proportion of harvest volume. In this way, the microorganisms remain in the fermentor to convert more nitrate into nitrite. In addition, this can provide more time for nitrate to be converted into nitrite to ensure that the product contains the desired concentration of nitrite. In addition, when calculating the volume of the fermentation source material harvested at regular time intervals in step 306, the conversion rate of nitrate to nitrite is also taken into account.

[0069] In step 307, the feed rate is determined from the harvest volume removed and the harvest time. In some embodiments, the feed rate is calculated so that the volume removed is replaced at the end of the time interval. In some embodiments, such a feed rate is calculated using the following formula:

[0070]

[0071] where Feed Rate (gallons / minute) is the rate of fresh feed required to replenish the volume of harvest, VHarvest (gallons / hour) is the volume of harvest per unit time, and

[0072] Harvest Time (hours) is the length of time required between harvests.

[0073] In some embodiments, the regular time intervals for harvesting the fermented liquid can be adjusted to be longer to protect downstream process equipment. In some embodiments, the required time intervals can be 30 minutes to 2 hours. In some embodiments, the harvest can be removed every 1 hour to 1.5 hours. The longer the time interval, the larger the harvest volume to be removed. As the fermented liquid is harvested, fresh source material liquid is simultaneously fed to the fermentor tank to replace the removed portion of the fermented liquid within a certain period of time.

[0074] In step 308, in some embodiments, concentration measurement can be carried out during the fermentation process to predict when nitrate is fully converted into nitrite. Once the nitrate of desired concentration has been converted into nitrite in the source material liquid, the first harvest is taken out. In some embodiments, nitrite concentration can be allowed to reach the stable state of the desired concentration range before taking out the first harvest. Afterwards, results can be carried out at regular time intervals. Meanwhile, fresh source material liquid is added to replace the liquid of results.

[0075] As described above, the rate at which fresh material is added is lower than the rate at which it is harvested. In some embodiments, the feed rate is adjusted based on the volume that must be replenished over a period of time (which is the same as the harvest volume). Because the microorganisms in the fermentor are already adapted and at their highest conversion rate, the additional volume of source material solution is fermented at the highest conversion rate of nitrate to nitrite.

[0076] In some embodiments, in order to maintain the fermentation at the highest conversion rate under given conditions, a challenge is to balance the rate at which the fermentation broth is removed with the rate at which the source material broth is added or the rate at which the microbial culture in the fermentor is replenished, or both. For example, to maximize nitrate conversion, a sufficient number of adapted microorganisms must be present in the fermentor. In some embodiments, about 7%-90% of the fermentation broth is removed from the fermentor. In some embodiments, about 20%-80% of the fermentation broth is removed from the fermentor. In some embodiments, about 40%-60% of the fermentation broth is removed from the fermentor. In some embodiments, the harvest volume can be less than 50% of the volume of the fermentor. In some embodiments, at least 30% of the microorganisms remain in the fermentor. In some embodiments, at least 40% of the microorganisms remain in the fermentor. In some embodiments, at least 50% of the microorganisms remain in the fermentor.

[0077] In some embodiments, the harvest volume can be kept constant, while the frequency of harvesting can be varied depending on the rate of conversion of nitrate to nitrite. When the nitrate is completely converted to nitrite, a predetermined amount can be harvested and fresh feed containing nitrate can be fed to the fermentor. The frequency of fermentation can be:

[0078]

[0079] In some embodiments, the fermented liquid is removed and replaced at a rate substantially equal to the growth rate of the microorganisms in the fermentor. The liquid is gradually fed to the fermentor at a defined feed rate. However, the removal of the liquid is completed as quickly as possible. In some embodiments, the removal is completed in 2-10 minutes, and in some embodiments, in 2-5 minutes. In this way, unconverted nitrates will not be collected from the feed liquid, which is undesirable. The removal can be completed quickly to reduce the loss of nitrates during harvest. In some embodiments, the rate of removal is 3-100 times faster than the rate fed into the fermentor. In some embodiments, the rate of removal is 5-50 times faster than the rate fed into the fermentor. In some embodiments, the rate of removal is 10-20 times faster than the rate fed into the fermentor. In some embodiments, the rate of removal is 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, or 35 times faster than the rate fed into the fermentor. For example, although a certain volume of fermented liquid is harvested in 5 minutes, the same volume of material source liquid is replenished within at least 1 hour.

[0080] Figure 4 Graph 400 shows exemplary feed and harvest rates to ensure that the volume of fresh feed equals the volume of harvest. After reaching the desired conversion level or nitrite concentration, a portion of the fermentation broth is removed for further processing. This removal occurs at desired intervals. In graph 400, removal occurs once per hour, but it can occur at more or less frequent intervals. Simultaneously, fresh source material is fed into the fermentor to replace the removed portion of the fermentation broth over time. As shown in graph 400, the harvest rate is faster than the feed rate.

[0081] Figure 5 An exemplary process flow diagram 500 for controlling a feedback loop for a continuous fermentation process is provided.

[0082] In some embodiments, under required feeding and harvesting rate, the volume of fresh feed equals the volume of harvest.Under this type of state, the nitrate and nitrite concentrations measured at regular time intervals are in desired concentrations.Based on the nitrate and nitrite concentrations measured at regular time intervals, feed rate, harvesting volume and harvesting time are adjusted.In some embodiments, process of the present disclosure includes a feedback loop to ensure that harvest includes nitrite and nitrate of desired concentration.For example, the feedback loop is used to ensure that all or substantially all nitrates have been converted, so harvest does not include or includes minimum nitrate and the nitrite of desired concentration, as discussed above.

[0083] If the nitrate or nitrite concentration is at the desired level 501, no adjustment is needed, and the set feed rate and harvesting rate 502 are maintained. If the nitrate concentration is too low 503, too much fermentation liquid will remain in the tank, and there will not be enough feed to enter the tank. In some embodiments, the concentration of nitrate can be zero, and microorganisms will begin to convert nitrite into nitrous oxide. To solve this problem, fresh nitrate can be provided to the fermentor tank. To achieve this, the feed rate 503a can be increased, and a larger volume of fermentation liquid 503b can be harvested at the same time interval, or the time interval 503c between harvests can be reduced. If the nitrite concentration is too low 504, there will not be enough fermentation liquid with active microorganisms in the tank, and too much feed will enter the tank. Microorganisms will convert nitrate into nitrite. To solve this problem, the feed rate 504a can be reduced, and a smaller volume of fermentation liquid 504b can be harvested at the same time interval, or the time interval 504c between harvests can be increased.

[0084] In some embodiments, the time to reach a desired conversion level can be monitored, and if this time changes, the feed rate of fresh source material liquor, the harvest time, or both can be changed to ensure the desired level of nitrate conversion to nitrite. In some embodiments, the conversion time may change due to changes in the number or activity level of the microorganisms.

[0085] In some embodiments, the conversion time may vary due to variations in the initial nitrate concentration in the fresh source material. In some embodiments, the harvested liquid is nitrate-free because all nitrate has been converted during the fermentation process.

[0086] refer to Figure 10, in some embodiments, an automated system for continuous fermentation of plant-based nitrates is provided. In some embodiments, as described above, when the source material liquid is inoculated with a bacterial culture capable of converting nitrate into nitrite, conversion data can be collected to monitor the conversion of nitrate to nitrite; for example, the conversion data includes changes in nitrate concentration and nitrite concentration in the source material liquid over time. In some embodiments, one or more sensors can be used to collect conversion data online or offline, such as collecting samples from a fermentation tank and testing the samples using HPLC. The conversion data can be transmitted to a computer system 1000, in particular a processor 342, which is programmed to perform one or more steps of the above method. For example, the processor can be programmed to receive conversion data; when the nitrite in the source material liquid reaches a desired concentration, a first harvest volume of the source material liquid is harvested at a harvest rate; and the harvest volume of the source material liquid is supplemented with a source material liquid containing additional nitrate at a feed rate, wherein the harvest rate is higher than the feed rate. In some embodiments, the processor can also be programmed to determine the conversion rate of nitrate to nitrite; calculate the conversion time (Tconversion) required to convert nitrate to nitrite based on the initial concentration of nitrate in the source material liquid and the conversion rate; calculate the harvest volume based on the total volume of the source material liquid and Tconversion; and calculate the feed rate based on the harvest volume and the harvest time.

[0087] For example, Figure 10 A schematic diagram of a typical processing architecture of a computer system 1000 is shown, which can be used in conjunction with the methods and systems of the present disclosure. A computer processing device 340 can be connected to a display 341 for graphical output. A processor 342 can be a computer processor 342 capable of executing software. A typical example can be a computer processor (e.g., or Processor 342 may be connected to memory 346, which may typically be volatile RAM memory for storing instructions and data while processor 342 is executing. Processor 342 may also be connected to storage device 348, which may be a non-volatile storage medium such as a hard drive, flash drive, tape drive, DVDROM, or similar device. Although not shown, computer processing device 340 typically includes various forms of input and output. I / O may include a network adapter, USB adapter, Bluetooth radio, mouse, keyboard, touchpad, display, touch screen, LED, vibration device, speaker, microphone, sensor, or any other input or output device used with computer processing device 340. Processor 342 may also be connected to other types of computer-readable media, including but not limited to electronic, optical, magnetic, or other storage or transmission devices, capable of providing a processor, such as processor 342, with computer-readable instructions. Various other forms of computer-readable media can transmit or carry instructions to a computer, including routers, private or public networks, or other transmission devices or channels, both wired and wireless. The instructions may include code from any computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0088] Program 349 can be a computer program or computer-readable code containing instructions and / or data and can be stored on storage device 348. The instructions can include code from any computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript. In general, processor 342 can load some or all of the instructions and / or data of program 349 into memory 346 for execution. Program 349 can be any computer program or process, including, but not limited to, a web browser, a browser application, an address registration process, an application, or any other computer application or process. Program 349 can include various instructions and subroutines that, when loaded into memory 346 and executed by processor 342, cause processor 342 to perform various operations, some or all of which may implement the methods for managing medical care disclosed herein. Program 349 can be stored on any type of non-transitory computer-readable medium, such as, but not limited to, a hard drive, a removable drive, a CD, a DVD, or any other type of computer-readable medium.

[0089] Further details of the present disclosure are illustrated by the following non-limiting examples. These examples are provided to fully disclose and describe to those skilled in the art how to make and use the compositions and methods of the present disclosure and are not intended to limit the scope of what the inventors consider to be their disclosure.

[0090] Example 1 - Celery Concentrate with 40,000 ppm Nitrates (Dry Basis)

[0091] Figure 6 600 is a graph showing the continuous conversion of nitrate to nitrite using a starting material containing 40,000 ppm nitrate (dry basis) according to some embodiments of the present disclosure. Nitrite concentration 601, nitrate concentration 602, and feed rate (gallons / minute) 603 are shown. After reaching the optimal conversion level, a portion of the fermentation broth is withdrawn for other processing. Simultaneously, fresh source material liquid is fed to the fermentor to replace the withdrawn portion of the fermentation broth over time.

[0092] Graph 600 shows that a 45 Brix celery concentrate having 40,000 ppm nitrate on a dry basis (soluble solids) was diluted to 7 Brix with water and the pH was adjusted to 4.8-7.5 using food grade sodium hydroxide. The diluted celery juice (3,500 gallons) was processed through an ultra-high temperature pasteurizer and fed to a fermenter. An actively growing culture of Staphylococcus carnosus in logarithmic growth phase was used to inoculate the celery juice at 37°C. Nitrate and nitrite were measured every hour to obtain the percent conversion of nitrate to nitrite. The nitrite concentration was plotted over time. The time at which nitrate was completely converted to nitrite was calculated by fitting the curve with the highest R 2 The conversion rate of nitrate to nitrite in the last hour (maximum rate) was determined to be approximately 20,000 ppm nitrate / hour (dry basis). By dividing 40,000 ppm nitrate by 20,000 ppm / hour, the time required for complete conversion at a conversion rate of 20,000 ppm / hour was determined to be 2 hours. The harvest volume was determined by dividing the fermentation volume by 2 hours, resulting in 1,750 gallons harvested per hour. The feed rate was determined by dividing 1,750 gallons by 60 minutes, which is 29 gallons / minute. Harvesting is accomplished at a faster rate, preferably in excess of 400 gallons / minute. Figure 6 As shown, a feed rate of 28-32 gallons per minute achieved a continuous process of approximately 95 hours. Approximately 90 harvests of 1,750 gallons of fermented celery juice produced 166,000 gallons of product, which would otherwise require 45 batch fermentations of 5 hours each (e.g., Figure 7 shown).

[0093] Figure 7is a graph 700 showing nitrate and nitrite (dry basis) concentrations over time in a batch fermentation using a starting material containing approximately 40,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure. Nitrite concentration 701 and nitrate concentration 702 are shown.

[0094] Example 2 - Celery Concentrate with 70,000 ppm Nitrates (Dry Basis)

[0095] Figure 8 800 is a graph showing the continuous conversion of nitrate to nitrite using a starting material containing 70,000 ppm nitrate (dry basis) according to some embodiments of the present disclosure. Nitrite concentration 801, nitrate concentration 802, and feed rate (gallons / minute) 803 are shown. After reaching the optimal conversion level, a portion of the fermentation broth is withdrawn for other processing. Simultaneously, fresh source material liquid is fed to the fermentor to replace the withdrawn portion of the fermentation broth over time.

[0096] Graph 800 shows that a 45 Brix celery concentrate having 70,000 ppm nitrate on a dry basis (soluble solids) was diluted to 7 Brix with water and the pH was adjusted to 4.8-7.5 using food grade sodium hydroxide. The diluted celery juice (3,500 gallons) was processed through an ultra-high temperature pasteurizer and fed to a fermenter. An actively growing culture of Staphylococcus carnosus in logarithmic growth phase was used to inoculate the celery juice at 37°C. Nitrate and nitrite were measured hourly to obtain the percent conversion of nitrate to nitrite. The nitrite concentration was plotted over time. The time at which nitrate was completely converted to nitrite was calculated by fitting the curve with the highest R 2 The conversion rate of nitrate to nitrite in the last hour (maximum rate) was determined to be approximately 23,500 ppm nitrate / hour (dry basis). By dividing 70,000 ppm nitrate by 23,500 ppm / hour, it was determined that the time required for complete conversion at a conversion rate of 23,500 ppm / hour was approximately 3 hours. The harvest volume was determined by dividing the fermentation volume by 3 hours, resulting in 1,167 gallons harvested per hour. Harvesting was performed every 1.5 hours (90 minutes) to ensure that downstream processing equipment was not adversely affected. Therefore, at 90 minute intervals, the harvest volume was set to 1,750 gallons. The feed rate was determined by dividing 1750 gallons by 90 minutes, which is approximately 20 gallons / minute. Harvesting is accomplished at a faster rate, preferably in excess of 400 gallons / minute. As Figure 8As shown, a feed rate of 18-21 gallons per minute achieves a continuous process of approximately 46 hours. Approximately 26 harvests of 1,750 gallons of fermented celery juice produce 45,500 gallons of product, which would otherwise require 13 batch fermentations of 7.5 hours each (e.g., Figure 9 shown).

[0097] Figure 9 is a graph 900 showing nitrate and nitrite (dry basis) concentrations over time in a batch fermentation using a starting material containing approximately 70,000 ppm nitrate (dry basis), according to some embodiments of the present disclosure. Nitrite concentration 901 and nitrate concentration 902 are shown.

[0098] The curing agent produced by the method of the present invention can include a nitrite amount for curing various meats, such as beef, pork, turkey, chicken, products of such meats, and seafood and seafood products. In some embodiments, the curing agent of the present invention can be used with organic or all-natural meats, livestock and poultry, or seafood. The curing agent of the present invention has a nitrite concentration sufficient to curing various meats or seafood products. For example, the curing agent of the present invention can have a nitrite concentration of at least 100ppm, at least 1,000ppm, at least 10,000ppm, or at least 20,000ppm. In some embodiments, the concentration of nitrite can be 20,000ppm-140,000ppm. The curing agent of the present invention can be combined with other meat curing products (such as sea salt). The curing agent of the present invention can be in liquid or powder form.

[0099] In some embodiments, a method of preserving meat, livestock, or seafood products comprises contacting the meat or seafood product to be preserved with a curing agent of the present disclosure. In some embodiments, a cured meat or seafood product is provided, wherein such product has been contacted with a curing agent of the present disclosure.

[0100] In view of the foregoing description, many modifications and alternative embodiments of the present disclosure will be apparent to those skilled in the art. Therefore, the description is interpreted as illustrative only, and its purpose is to teach those skilled in the art the best way to implement the present disclosure. Without departing from the spirit of the present disclosure, the details of the structure may be substantially changed, and exclusive use is reserved for all modifications that fall within the scope of the appended claims. In this specification, the embodiments have been described in a manner that allows for a clear and concise description to be written, but it is intended and should be understood that the embodiments can be combined or separated in various ways without departing from the present disclosure. The present disclosure shall be limited only to the scope required by the appended claims and the applicable legal rules.

[0101] It will also be understood that the following claims are intended to encompass all generic and subordinate features of the disclosure described herein, and that all statements of the scope of the disclosure, as a matter of language, might be said to fall therein.

Claims

1. A method for continuously producing a pickling agent, the method comprising: inoculating a fluid of source material containing nitrate with a bacterial culture capable of converting nitrate to nitrite; When the nitrite in the source material liquid reaches a desired concentration, harvesting a first harvest volume of the source material liquid at a harvest rate; and The first harvest volume of source material liquid is replenished with source material liquid comprising additional nitrate at a feed rate, wherein the harvest rate is higher than the feed rate.

2. The method of claim 1, further comprising: Determine the conversion rate of nitrate to nitrite; Calculating T conversion based on the initial concentration and conversion rate of nitrate in the source material liquid, wherein T is the required conversion time for nitrate to be converted into nitrite; Calculating a first harvest volume based on the total volume of the source material liquid and the T conversion; and The feed rate is calculated based on the first harvest volume and harvest time.

3. The method of claim 2, further comprising: One or more additional harvest volumes of the source material fluid having the desired nitrite concentration are harvested at regular time intervals equal to the harvest time.

4. The method according to claim 3, wherein: If the nitrite concentration in the one or more additional harvest volumes is lower than the desired nitrite concentration, the feed rate is decreased, the one or more additional harvest volumes are reduced, or the harvest time is increased.

5. The method according to any one of claims 1 to 4, wherein The nitrate in the source material liquid is a plant-based nitrate.

6. The method of any one of claims 1 to 4, further comprising: The initial concentration of nitrate in the source material solution is adjusted.

7. The method of claim 2, wherein: The conversion rate of nitrate to nitrite is the maximum conversion rate of nitrate to nitrite by the bacterial culture.

8. The method of claim 7, further comprising: One or more of the first harvest volume, the harvest rate, and the feed rate are adjusted to maintain maximum conversion of nitrate to nitrite.

9. The method of any one of claims 1 to 4, further comprising: The conversion rate of nitrate to nitrite is determined and allowed to reach a maximum conversion rate of nitrate to nitrite by the bacterial culture before harvesting the first harvest volume.

10. The method according to any one of claims 1 to 4, wherein The first harvest volume is 20%-80% of the initial volume of the source material liquid.

11. The method according to any one of claims 1 to 4, wherein The harvest rate is 3-100 times faster than the feed rate.

12. The method according to any one of claims 1 to 4, wherein The nitrate concentration of the source material liquid is at least 100 ppm.

13. The method according to any one of claims 1 to 4, wherein The source material liquid has a Brix measurement of 1-12.

14. The method according to any one of claims 1 to 4, wherein The desired nitrite concentration is reached when at least 85% of the nitrate has been converted to nitrite.

15. The method according to any one of claims 2 to 4, wherein The harvest time is 30 minutes to 2 hours.

16. A system comprising: a fermenter configured to contain a liquid source material comprising plant-based nitrates, the fermenter having an inlet configured to provide the liquid source material to the fermenter and an outlet configured to withdraw the liquid source material from the fermenter; a processor programmed to receive conversion data to monitor conversion of nitrate to nitrite when the source material fluid is inoculated with a bacterial culture capable of converting nitrate to nitrite; When the nitrite in the source material liquid reaches a desired concentration, harvesting the first harvest volume of the source material liquid at a harvest rate; and The first harvested volume of source material fluid is replenished with source material fluid comprising additional nitrate at a feed rate, wherein the harvest rate is higher than the feed rate.

17. The system of claim 16, wherein: The processor is further programmed to: Determine the conversion rate of nitrate to nitrite; Calculating T conversion based on the initial concentration and conversion rate of nitrate in the source material liquid, wherein T conversion is the required conversion time for nitrate to be converted into nitrite; Calculating a first harvest volume based on the total volume of the source material liquid and the T conversion; and The feed rate is calculated based on the first harvest volume and harvest time.

18. The system of claim 16, wherein: If the nitrite concentration in the one or more additional harvest volumes is below the desired nitrite concentration, the processor is further programmed to cause the feed rate to be decreased, the one or more additional harvest volumes to be reduced, or the harvest time to be increased.

19. The system of claim 16, wherein: The nitrate in the source material liquid is a plant-based nitrate.

20. The system of claim 17, wherein: The conversion rate of nitrate to nitrite is the maximum conversion rate of nitrate to nitrite by the bacterial culture.

21. The system of claim 20, wherein: The processor is further programmed to adjust one or more of the first harvest volume, the harvest rate, and the feed rate to maintain maximum conversion of nitrate to nitrite.

22. The system of any one of claims 16 to 21, wherein: The processor is further programmed to determine the conversion rate of nitrate to nitrite and allow the conversion rate to become a maximum conversion rate of nitrate to nitrite by the bacterial culture before harvesting the first harvest volume.

23. The system of any one of claims 16 to 21, wherein: The first harvest volume of the fermented source material liquid is 20% to 80% of the initial volume of the source material liquid.

24. The system of any one of claims 16 to 21, wherein: The harvest rate is 3-100 times faster than the feed rate.

25. The system of any one of claims 16 to 21, wherein: The nitrate concentration of the source material liquid is at least 100 ppm.

26. The system of any one of claims 16 to 21, wherein: The source material liquid has a Brix measurement of 1-12.

27. The system of any one of claims 16 to 21, wherein: The desired nitrite concentration is reached when at least 85% of the nitrate has been converted to nitrite.

28. The system of any one of claims 17-18, wherein: The harvest time is 30 minutes to 2 hours.

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