Method for microbial sampling, and method for applying global sampling to food items

Abstract

Certain aspects of this disclosure relate to methods and apparatus for microbial sampling of food. For example, a method may include providing at least one aggregating sampler at one or more sampling locations and sampling a production batch of product or other food items, such as meat, using the at least one aggregating sampler to create one or more samples that comprise a microbial sample. Certain aspects of this disclosure relate to methods and apparatus for microbial sampling of food. For example, an apparatus, such as a microbial aggregating sampler, may include a cover having a microbial sampling material with a pocket formed in the cover to receive a limb or tool for handling the cover.

Description

49 METHOD FOR MICROBIAL SAMPLING, AND METHOD FOR APPLYING GLOBAL SAMPLING TO FOOD ITEMS CROSS-REFERENCED TO RELATED APPLICATION(S)

[001] This request claims priority for US Request No. 16 / 057,137, filed August 7, 2018, which claims priority to and benefit of U.S. Provisional Patent Application Serial No. 62 / 656,164, filed April 11, 2018, U.S. Provisional Patent Application Serial No. 62 / 589,755, filed November 22, 2017, and U.S. Provisional Patent Application Serial No. 62 / 543,220, filed August 9, 2017, all assigned to the assignee hereof and hereby expressly incorporated herein by reference. FUNDAMENTALS Dissemination Field

[002] This disclosure relates generally to improving the food safety of ready-to-eat products and other food items and to providing process validation and, more particularly, to methods and apparatus for microbial sampling of food items and other materials. Description of the Related Technique

[003] The microbial testing process has undergone tremendous changes in recent years. Traditional plating techniques for enumeration and detection have given way to faster and more specific antibody-based and molecular biology techniques. These newer techniques may not require time for colonies to form, but they may generally require enrichment culture to collect enough of the target organism and remove interference.

[004] In the ready-to-consume products industry, millions of dollars are spent collecting random samples attempting to demonstrate product safety in an effort to meet customer demands. Petition 870240102506, dated 02 / 12 / 2024, page 9 / 95 / 49, refers to an ever-increasing number of tests. These efforts may be technically and statistically flawed and may not meet expectations of ensuring food safety. In particular, random samples are too small to represent production batches of material. Production batches of material are too heterogeneous for random sampling to be descriptive of the production batch. Furthermore, results arrive too slowly to make decisions without sacrificing quality. Pathogen levels are generally so low that the occasionally positive sample reflects the baseline that is always present rather than a deviation from the norm. The ready-to-eat products industry can benefit from effective cross-contamination testing and control. It can also benefit from an effective measure of process effectiveness and deviation in processing.Increasing the effectiveness of raw material testing can help improve food safety practices.

[005] Thus, as the demand for microbial sampling continues to increase, there is a desire for further improvements in sampling techniques and technology. Preferably, these improvements should be applicable to other related technologies and the methods and devices that employ these technologies. BRIEF SUMMARY

[006] The systems, methods, and devices of the disclosure each have diverse aspects, none of which is solely responsible for its desirable attributes. Without limiting the scope of this disclosure as expressed by the claims that follow, some features will now be discussed briefly. After considering this discussion and particularly after reading the section entitled “Detailed Description,” one will understand how the features of this disclosure provide advantages that include improved microbial sampling of food and other products.

[007] Certain aspects provide a method for sampling Petition 870240102506, dated 02 / 12 / 2024, page 10 / 95 / 49 microbial of food and other products. The method generally includes obtaining a microbial sample from one or more items, extracting the microorganisms from the microbial sample, concentrating the microorganisms, cleaning the microorganisms, recording the relative presence of microorganisms and any potential pathogens, aggregating this information from a microorganism record within a microorganism report, confirming the microorganism record and reporting the microorganism report.

[008] Certain aspects provide a method for microbial sampling of food. The method generally includes providing at least one aggregating sampler at one or more sampling locations and sampling, using at least one aggregating sampler, a production batch from the production, creating one or more samples that comprise a microbial sample.

[009] The aspects generally include methods, apparatus, systems, computer-readable media and processing systems, as substantially described herein with reference to and as illustrated by the accompanying drawings.

[0010] For the fulfillment of the preceding and related purposes, the one or more aspects comprise the features described below in full and particularly highlighted in the claims. The following description and the accompanying drawings detail certain illustrative features of the one or more aspects. These features are indicative, however, of only some of the various ways in which the principles of various aspects may be employed, and this description is intended to include all such aspects and their equivalents. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order for the features mentioned above in this disclosure to be understood in detail, a more specific description is provided, Petition 870240102506, dated 02 / 12 / 2024, page 11 / 95 / 49, briefly summarized above, can be understood by reference to the aspects, some of which are illustrated in the attached drawings. However, it should be mentioned that the attached drawings illustrate only certain typical aspects of this disclosure and, therefore, should not be considered limiting to its scope, so that the description may admit other equally effective aspects.

[0012] FIG. 1 illustrates exemplary operations for microbial sampling, in accordance with aspects of this disclosure.

[0013] FIG. 2 illustrates exemplary operations for applying aggregation sampling, in accordance with aspects of this disclosure.

[0014] FIG. 3 illustrates Table 1 which includes exemplary treatments and results for meat sampling, according to aspects of this disclosure.

[0015] FIG. 4 illustrates Table 2 which includes exemplary treatments and results for sampling of leafy vegetables, according to aspects of this publication.

[0016] FIG. 5 illustrates an aggregating sampler that includes a bag in accordance with aspects of the present disclosure.

[0017] FIG. 6 illustrates an aggregating sampler that includes a bag extending through a cover in accordance with aspects of the present disclosure.

[0018] FIG. 7 illustrates an aggregating sampler with at least one convex surface according to aspects of the present disclosure.

[0019] FIG. 8 illustrates an aggregating sampler that includes at least one concave surface in accordance with aspects of the present disclosure.

[0020] FIG. 9 illustrates an aggregating sampler that includes a curved shape in accordance with aspects of the present disclosure.

[0021] FIG. 10 illustrates an aggregating sampler that includes a borehole. Petition 870240102506, dated 02 / 12 / 2024, p. 12 / 95 / 49 or loop in accordance with aspects of this disclosure.

[0022] FIG. 11 illustrates an aggregating sampler that has the shape of a mitten or glove for one hand according to aspects of the present disclosure.

[0023] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is considered that the elements disclosed in one aspect may be beneficially used in the other aspects without specific citation. DETAILED DESCRIPTION

[0024] Aspects of this disclosure provide apparatus, methods, and / or systems for automated and semi-automated microbial sampling of food and other materials. Other materials may be as diverse as water or air streams. More commonly, these will include food-related products such as animal feed, medical materials, or dietary supplements where microbial testing is required to confirm hygienic operation. Sampling may be active, as mediated by material flow, or operator-mediated by applying the sampler to a surface. Sampling may also be more passive and rely on passive contact or gravity sedimentation.

[0025] The following description provides examples and is not limiting the scope, applicability, or examples presented in the claims. Changes may be made to the function and arrangement of elements discussed without departing from the scope of the disclosure. Various examples may omit, replace, or add various procedures or components as appropriate. For example, the methods described may be performed in a different order than that described, and various steps may be added, omitted, or combined. Also, traits described with respect to some examples may be combined in some other examples. For example, a Petition 870240102506, dated 02 / 12 / 2024, p. 13 / 95 / 49. An apparatus may be implemented or a method may be practiced using any number of the aspects presented herein. Furthermore, the scope of the disclosure is intended to encompass such an apparatus or method practiced using a different structure, functionality, or structure and functionality in addition to or other than the various aspects of the disclosure presented herein. It should be understood that any aspect of the disclosure disclosed herein may be embodied by one or more elements of a claim. The word “exemplary” is used herein to mean “serving as an example, case, or illustration.” Any aspect described herein as “exemplary” is not necessarily to be interpreted as preferred or advantageous in relation to other aspects.

[0026] In one embodiment, devices are mounted and connected to a rapid reporting system to provide more representative sampling and faster analysis of a food product or other material. In another embodiment, a similar system provides robotic sampling of a field crop and release of results with a mobile laboratory. Both embodiments may include a two-stage screening system for speed and economy, but a conventional testing approach may be considered to take advantage of the sampling improvements.

[0027] There has been little evolution in sampling and sample preparation with regard to submission to these advanced and rapid analytical techniques. One technique is to collect periodic samples or random picks from a batch. This sample is extracted by homogenization or stomaching and then a portion is directly analyzed when high populations are expected or enriched before analysis. Liquid samples, and particularly water samples, can be filtered to allow analysis of larger samples. For special purposes, but generally not for routine analysis, liquid samples can be concentrated by centrifugation to pelletize. Petition 870240102506, dated 02 / 12 / 2024, page 14 / 95 / 49 microorganisms. There are statistically sound sampling plans as recommended by academic or international organizations, but these sampling plans are rarely practical and are often prohibitively expensive. Such sampling plans in use based on periodic sampling or random sampling have a very low data density. Furthermore, these sampling plans inherently have sampling biases due to batch geometry, where portions of the batch are essentially not sampled, and the heterogeneous nature of microorganism distribution.

[0028] Some microbial tests may assume that test organisms are uniformly distributed, allowing a random sample to be representative of the whole. This assumption fails to represent non-homogeneous distributions in aggregate and requires many samples to characterize the microbial population of a batch. Test inspection and release based on random samples can be flawed to the degree that random sampling may inherently fail to detect significant contamination. Furthermore, sample size limits the detection threshold to levels that may be orders of magnitude above the baseline rate and the level where risk becomes imminent.On the other hand, microbial proliferation during enrichment culture occurs under conditions selected to favor the growth of potential pathogens, which often have no relation to commercial storage conditions and therefore generates distorted perceptions of the risk of microbial growth.

[0029] One or more of the modalities as disclosed herein may address these shortcomings of current sampling and testing practices and provide a system for generating more meaningful, real-time or near real-time assessment at the site of microbial contamination risk. EXAMPLE OF AUTOMATED AND SEMI-AUTOMATED MICROBIAL SAMPLING OF FOOD AND OTHER MATERIALS Petition 870240102506, dated 02 / 12 / 2024, page 15 / 95 / 49

[0030] According to one or more aspects of the embodiments disclosed herein, automated and semi-automated microbial sampling of food and other materials is provided. For example, devices can be mounted and connected to a rapid reporting system to provide more representative sampling and faster analysis of a food product. In another case, a system can provide robotic sampling of a field crop and can release results with a mobile laboratory. Both examples can include a two-stage screening system that can provide speed and economy. Although less advantageous, it is reasonable to take advantage of enhanced sampling with conventional enrichment and detection systems.

[0031] In one or more cases, a microbial sampling method and system includes providing a sampling plate, such as a MicroTally Plate, which is then used with a continuous sampling device (CSD) to collect biological agents. The CSD can automatically change the sampling plate between samples. The sampling plate is then used for analysis. In particular, target bacteria can be removed from the sampling medium. The target bacteria samples are cleaned and concentrated using a treater and made suitable for analysis. The treated sample is then analyzed using molecular or biochemical methods, and target agents are detected with acceptable accuracy and sensitivity. A cloud-based data reporting system with user-appropriate dashboards to report actionable information and facilitate timely decision-making can also be provided.

[0032] For example, FIG. 1 illustrates specific operations 100 for microbial sampling, according to aspects of this disclosure.

[0033] Specifically, operations 100 begin, in block 102, with obtaining a microbial sample of one or more food items which may include, for example, produce, meat and other items. Petition 870240102506, dated 02 / 12 / 2024, page 16 / 95 / 49 food or other materials. In block 104, operations 100 include extracting microorganisms from the microbial sample. In block 106, operations 100 include concentrating the microorganisms. Operations 100, in block 108, include cleaning the microorganisms. Furthermore, operations 100 include, in block 110, recording the relative presence of microorganisms and any potential pathogens. Operations 100 also include, in block 112, aggregating this information from a microorganism record into a microorganism report. In block 114, operations include confirming the microorganism record. Operations may also include, in block 116, reporting the microorganism report from the microorganism registry.

[0034] In one or more cases, sampling may involve using a sampling plate or swab that collects a sample and is stomached in a 300-micron partitioned bag with 200 ml of elution buffer. Sample concentration and cleanup may include siphoning elution buffer and trapping organisms through a patented sequential filter, and targets may be deposited onto a 0.22-micron PC filter. Targets are analyzed from the filter, which may include, for example, purified DNA, and qPCR is conducted for index elements including pathogen intensity, enteric status, and / or positive control. Cloud-based reporting, based on, for example, Ignition and SQL databases, can be provided with appropriate user dashboards to provide timely actionable information.

[0035] Depending on one or more cases, pathogens may be confirmed by resampling and enrichment procedures, by resampling and performing definitive pathogen tests, or by performing confirmatory tests on the original DNA, depending on regulatory guidance. In one or more cases, a cassette and cartridge system may be implemented to improve the sampling process. Although not required, the Petition 870240102506, dated 02 / 12 / 2024, page 17 / 95 / 49: Elimination of the use of a digester and partitioned bag may be provided in one or more cases. An agglutination collector may be provided to replace the PC membrane filter, which can help improve sample distribution to the detector and potentially eliminate the need for DNA purification. The use of ribosomal RNA may become a new standard for one or more such cases. In one or more cases, a detector may use flow amplification and laboratory-on-a-chip technology to further reduce detection times and costs.

[0036] To better address all the shortcomings in current practice, numerous improved elements can be combined. Taken individually, each improvement addresses some of the shortcomings and produces some advantage. Skipping the entire path to a complete solution may be beyond the sophistication of some industries, so grouping intermediate steps is considered for each element. For the initial discussion, the elements under consideration include Sampling, Extraction, Concentration, Cleaning, Screening, Detection, Second-Stage Sampling of Suspect Batches, Reporting, and Information Recording. Although not as desirable, a more conventional enrichment can be used instead of Concentration. After such enrichment, any number of detection systems can be used to detect the presence or absence of a target organism. Each of these elements is discussed below.

[0037] Thus, the sampling period for information may be driven by the needs of the business class, but is not limited to this. Products with a short shelf life may justify greater speed. Valuable goods such as meat products will require more testing to limit exposure when a problem is expected or anticipated. These factors will impact the degree to which a fully automated solution is implemented or, conversely, when a system more similar to current practices is used to obtain some of the benefits of sampling. Petition 870240102506, dated 02 / 12 / 2024, page 18 / 95 / 49, improved. Sampling Examples

[0038] There are practical limits to the amount of product that can be sampled by conventional means. Without heroic efforts, samples are limited to the small fraction of a pound (usually 150 grams or less, but some laboratories are routinely testing as much as 300 grams) which leads to operating curves for c=0 acceptance that have an inflection at about 1 CFU / pound. The net effect of such sampling and testing is the erroneous belief that the worst lots are detected when 1 positive is found in many hundreds or thousands of samples. Unfortunately, this test is far removed from the range of interest; it is little more than a random selection of lots to be rejected.

[0039] Manual plate sampling can increase the effective sampling weight 20 or 30 times, which is enough to shift the operating curve about an order of magnitude to the left by about 0.1 CFU / pound if the same inspection criterion c=0 is used. Similarly, if a continuous sampling method is used, the effective sample can be increased 200 to 300 times, producing an additional order of magnitude in LOD at about 0.01 CFU / pound.

[0040] The surface area of ​​an aggregating sampler provides advantages beyond material sampling when greater sensitivity is desired for surface and fluid flow sampling. Sampling water and air streams are two examples where flow sampling is advantageous. Surface area is also applicable to sampling surfaces where topical contamination is of concern.

[0041] The use of a sampler has the advantage of being non-destructive and can produce exceptional efficiencies. However, the real advantage results when this LOD is replaced by statistical process control with a two-stage acceptance criterion where deviations from the normal are detected. Petition 870240102506, dated 02 / 12 / 2024, page 19 / 95 / 49 as opposed to randomly selecting batches for reallocation. This concept is discussed more fully below when this debate returns to screening detection. This is an important distinction when the goal is to improve the microbial safety of a product or material. This line of argument also allows for faster detection of many cells rather than waiting for one cell to develop into many cells. With the recognition of the power of the larger and more effective sampling procedure, there may be a need to expand the range of tools to apply this technology to a wider array of products with alternative geometries and increased levels of automation. For example, the geometry could be altered to allow sampling of a powder stream through a tube with a circular geometry where a segment of the tube is swapped between batches.The pipe section would be lined with sampling material or, better yet, include baffles maximizing product contact with the sampling surfaces. Alternatively, vertical chutes could be provided below the bag fillers to sample product just before bagging in a bag forming, filling, and sealing machine.

[0042] A microbial sample may include, for example, nonwoven fabric, various microfiber materials, sponges and / or any absorbent sheet material. Polypropylene or polyethylene nonwoven fabric is of particular mention since these materials are left in contact with food and therefore have very low extractables that would otherwise contaminate the material stream under examination.

[0043] Additionally, the usefulness of these sampling approaches can be increased with automation. A feed cartridge can be used to release multiple plates to the sampling location at once. This cartridge would be placed in the line after sanitization has been completed on all preparations, protecting the drive mechanisms from the aggressive cleaning process. Petition 870240102506, dated 02 / 12 / 2024, page 20 / 95 / 49

[0044] Similarly, a cassette case can be loaded to collect individual pieces of sampling material. Both the cartridge and the cassette are engineered to advance the sampling material when appropriate (e.g., when a batch is completed, when a load is moved, etc.). The driving force to advance the sampling plates can be provided by a motor or supplied by a hand crank or cable or by an operator depending on the characteristics of the operation. Both the cassette and the cartridge can be designed to protect the microbial integrity of the contents. Cartridges can prevent microbial growth after sampling, external contamination, and cross-contamination between batches.

[0045] The sampling material can be mounted on an inert reinforcing material to facilitate sanitary placement of the sampling plates. Alternatively, the sampling material plates can be separated by short gaps of inert material to ensure that used sampling materials do not contaminate other plates.

[0046] The usual design parameters for process equipment can be applied to these devices, including, for example, an aggregating sampler. For example, thick 316 stainless steel is a suitable material. When the sampling device is not in place, the location can be passive, such as a dead plate where the product passes through without damage or obstruction. It can also be sanitaryly designed from the outset so that it is easily cleaned.

[0047] In one case, for this automation to have maximum benefit, the cartridges can carry information regarding the sample they contain. If the sample is used in a manual mode, an electronic transfer of this information along with the sample is also advantageous to avoid human error and speed up the flow of information. This information transfer can occur via the cloud using barcodes, a base Petition 870240102506, dated 02 / 12 / 2024, page 21 / 95 / 49 regarding data and location information.

[0048] Another class of geometries is needed to extend the power of this sampling to agricultural commodities in the field. Such sampling has utility beyond testing for human pathogens, as it can be used to test for plant pathogens that can reduce crop productivity. For example, early detection of mold spores motivates the early harvesting of a spinach field. Detection of rust in a wheat or corn field would motivate the use of a disease control measure in the affected field before the rust destroys the crop.

[0049] Depending on the field setting, an octopus tentacle configuration may be the appropriate geometry where filaments of sampling material are sliding across the crop surface. These filaments can be ropes or frayed strips of material depending on what provides the most effective contact. These tentacles can be brought into contact with the crop by various mechanisms including robots, tractors, hand-held or drones. It is most important that the altitude is kept constant to allow contact while minimizing damage to the crop. To increase sampling effectiveness, it may be advantageous to absorb moisture below these tentacles or install a vacuum or suction mechanism.

[0050] For crops that have a more uniform top surface, such as baby vegetables or spinach, sheet materials may be more effective since the surfaces of new leaves facing upwards are omitted. For these crops, air-based sampling with suction or electrostatic sampling, which increases the efficiency of microbial sampling, presents an interesting alternative.

[0051] For manual sampling, forming the sampling material into bags, mitts, or gloves can facilitate use. Ease of use will lead to greater adherence to the sampling protocol. In a manual mode, the Petition 870240102506, dated 02 / 12 / 2024, page 22 / 95 / 49. The duration of contact is a factor in determining sampling effectiveness. Typical durations are minutes. Durations of 2 to 5 minutes will work for most applications.

[0052] FIG. 2 illustrates example operations 200 for applying aggregation sampling, in accordance with aspects of this disclosure. Specifically, operations 200 begin, in block 202, with the provision of at least one aggregation sampler at one or more sampling locations. Additionally, operations 200 include, in block 204, sampling, using at least one aggregation sampler, a batch of produce, creating one or more samples that comprise a microbial sampling. One or more samples may be configured to be processed to indicate whether pathogens are present on no more than a normal basis. The one or more sampling locations may include at least one in a field, at harvest, immediately after unloading or cutting, in a washing system, or after the washing system.In one or more cases, additional operations may be included, such as, for example, evaluation, use of an aggregating sampler, or a level of cross-contamination control to validate or verify a washing process.

[0053] In one or more cases, an aggregation sampler may be provided that sufficiently samples a production batch of ready-to-eat products to confirm that pathogens are present on no more than the normal basis. In one or more cases, an aggregation sampler may assess the level of cross-contamination control to validate or verify a washing process.

[0054] In one or more cases, an aggregating sampler and a sampling location for the aggregating sampler may be provided. Additionally, sampling by the aggregating sampler may be provided to generate one or more desired samples. Analysis of one or more samples and interpretation of the analysis results may also be provided. These elements Petition 870240102506, dated 02 / 12 / 2024, p. 23 / 95 / 49, can be practiced individually or together to enhance food security.

[0055] The aggregating sampler may include a collection surface and an apparatus for holding and positioning the collection surface such that the collection surface contacts the product to be sampled for microorganisms or other targets. In one or more cases, a surface with a sampling efficiency that allows for an increased effective sample size when a production batch of the product is sampled may be provided. For example, two hours of production of a leafy vegetable product may be sampled with such a device. During the two hours a large quantity, for example 4,536 to 13,600 kg (10,000 to 30,000 lb), of product will have crossed the sampling surface. If the sampling device is at least 25% efficient as shown in laboratory-scale studies, the effective sample size may be 1,133 to 3,400 kg (2,500 to 7,500 lb).These sample sizes are enormous when compared to the few hundred grams of a sample collected normally.

[0056] In one or more cases, there are several factors to consider in the selection or design of an aggregating sampler. Initially, a sampling surface that is compatible with the product is provided. This typically means that the sampling surface is a food-grade material. For example, in one or more cases, a non-woven polyolefin cloth has proven effective. Another factor may include a design that allows for quick and safe replacement of the sampling surface. Yet another factor that may be included relates to any equipment left on the line being easily cleaned and incorporating a sanitary design. For example, food-grade stainless steel may be a material selected when implementing an aggregating sampler.

[0057] To test a product, there are several areas where sampling Petition 870240102506, dated 02 / 12 / 2024, page 24 / 95 / 49. Aggregate sampling can produce improvements in food safety. For example, some locations include: 1) in the field; 2) at harvest; 3) immediately after unloading or cutting; 4) in the washing system; or 5) after the washing system. In each area, there are particular locations that can be selected for aggregate sampling, but these will vary with the specific line configuration and the product to be sampled. Not all areas will be appropriate for all products. Selections can be guided by the desired information. Currently, random samples are frequently collected in all these areas, but these random samples are unable to represent the population under examination and such sampling is generally destructive.

[0058] According to one example, an aggregating sampler can be provided that can replace the current practice of collecting a sample picked in a field by cutting leaves. In particular, the aggregating sampler can be configured such that it can be carried by hand or mounted on a device designed to move through the field. The collection surface for sampling in the field can be divided to allow more conformity to the crop surface.

[0059] According to another example, during harvesting, an aggregating sampler can be placed on the combine harvester so that the product is sampled during the harvesting process. This approach would allow the matching of the harvested product with specific information. Although not required, the placement of the aggregating sampler can be done immediately after any classification is made in the field. For example, if stones are separated from the product by density classification, these stones do not need to cross the surface of the sampler.

[0060] In another example, right after unloading or cutting and before washing, the product may still contain flora found in the field. An aggregating sampler, placed in advance during the process, can sample Petition 870240102506, dated 02 / 12 / 2024, page 25 / 95 / 49 these organisms. Soon after cutting or chopping, the interior of some products will be exposed for the first time, allowing a more representative sample to be collected.

[0061] In a washing system, an aggregating sampler can collect a different type of sample according to one or more examples. This sampler can test the cross-contamination control of the washing system. The organisms collected will reflect the two most likely mechanisms of cross-contamination: water-mediated cross-contamination and product-to-product cross-contamination. An aggregating sampler, placed in the flow of the transported product, will be impinged by both water and product. To avoid excessive obstruction of the product flow, depending on the line design, the sampler can be placed at any angle from parallel to the product flow to completely perpendicular to the product flow. The sampler can also be a comb-type device with multiple collection probes between the product and the washing flow.The angle of attachment can affect the balance between water-mediated cross-contamination and observed product-to-product cross-contamination. In each case, this type of sampling can be used to validate cross-contamination control and the effectiveness of the washing solution.

[0062] Sampling in a washing system is a case where the sampling surface can be active on multiple surfaces, for example, on both sides or around in the case of the comb-type structure mentioned above. In one or more cases, it may be advantageous to laminate two sheets together with an impermeable coextruded adhesive layer to increase the bonding potential relative to the detachment potential, preventing flow through the sampling surface. Additionally, other advantages can be provided in other ways, such as increasing the thickness of the sampling material. In some cases, the design of Petition 870240102506, dated 02 / 12 / 2024, page 26 / 95 / 49 samplers in devices such as the filter housing may be provided. In other cases, placement on a sampling surface in an active area of ​​the washing system may be provided to obtain a complete measure of the potential for cross-contamination.

[0063] A sample can be collected after the washing system and will reflect a residual population. In this area there are several specific locations that can be considered depending on the specifics of the line. For example, these specific locations include just before the loading dryers, on a conveyor that would be used to lift the product for packaging, just before a bag scale or at the neck of a bag forming, filling and closing machine. This continuous in-line sampling can significantly increase sampling efficiency and provide more meaningful data than random finished product testing sampling.

[0064] With the samples collected, attention can be focused on the analysis and interpretation of results as discussed here. For example, in the specific product category, there are specific opportunities to be considered that can be provided with aggregate sampling using the aggregation sampler. The opportunities are provided in part due to the more representative nature of aggregate samples and the enormously increased numbers of organisms available in the samples compared to typical collected samples. These samples can be analyzed to provide multiple data channels depending on the detector technology employed. In one or more cases, the sample can be analyzed with metagenomics, allowing the entire population to be studied, producing a large, and in some cases maximal, amount of data that can be mined in various ways to gain knowledge and understanding of positive and negative deviations.This range of possibilities can be illustrated with several examples, but they are not limited to these. Petition 870240102506, dated 02 / 12 / 2024, p. 27 / 95 / 49

[0065] In one or more instances, samples collected in the field, at harvest, or just before washing can be used to assess the microbiological status of the raw material. From a food safety perspective, the focus has previously been on the presence or absence of pathogens. Unfortunately, such analyses based on random samples are unable to truly answer the question of whether pathogens are present due to their lack of sensitivity. Generally, pathogens are known to be present in very low numbers. These are ubiquitous organisms. A more appropriate question is whether these organisms are present in abnormal concentrations or without the usual competing organism.

[0066] In one or more examples, samples collected just before washing can be compared with samples collected in the washing system to directly measure cross-contamination using wild-type flora. Water samples may tend to have very low microbial loads in appropriately controlled washing systems even when cross-contamination is occurring. Wild-type flora can also be highly variable. However, using aggregating samplers at both locations can dampen noise and measure cross-contamination. Several metrics for cross-contamination can be considered based on the ratio of pre-wash sample results to those of the wash. Using more sophisticated analytical procedures, one can overcome the shortcomings of such metrics as aerobic plate count (APC), which would include many organisms that are not relevant for controlling pathogen cross-contamination.For example, spore-forming bacteria such as Bacillus will not be affected by washing and would only obscure any cross-contamination metric based on APC. However, with more focused data channels as provided by modern molecular techniques, better information can be obtained. Another aspect of this tool that can be provided is that... Petition 870240102506, dated 02 / 12 / 2024, page 28 / 95 / 49 statistical process control can be applied to detect deviations.

[0067] In one or more cases, samples taken after a washing process can provide information about the microbial populations in the finished product. These samples can provide a much more accurate assessment of the product's pathogen risk and better detection deviations. These samples can also be used to check for deviations in the normal flora.

[0068] In some cases, a ratio between pre-wash samples and post-wash samples can be used to assess the impact of the washing process. Aggregating samplers can reduce noise and, as with cross-contamination metrics, these ratios can be manipulated with statistical process control to look for deviations.

[0069] One or more of these examples can be distributed in near real-time because the aggregating samplers collected enough cells for concentration and direct analysis without enrichment. SAMPLING EXAMPLES: LABORATORY-SCALE EXAMINATION OF SAMPLING EFFICIENCY FOR RAW MEAT

[0070] According to one or more cases, an example from a laboratory-scale study demonstrates a sampling efficiency of 15 to 20% in relation to stomaching. The same also shows that the transfer is essentially instantaneous and that repeated contact collects more organisms. These observations confirm the intuitive assertion that continuous sampling will yield better information than random sampling.

[0071] For example, for a laboratory-scale experimental study, purchased beef stew is inoculated by immersion in a mixed culture of generic E. coli at ~105 CFU / ml. The E. coli is selected as benign and easy to enumerate. The beef stew is left to stand. Petition 870240102506, dated 02 / 12 / 2024, page 29 / 95 / 49 at room temperature for 30 minutes to allow adhesion. Then the six treatments listed in the table shown in FIG. 3 can be performed in 5 replicates. All sampling cloths can be cut in half, 30 cm by 20 cm (12 in by 8 in), to reduce sample requirements and facilitate experiment execution. The sampling cloth can be, for example, a MicroTally cloth but is not limited to this. The surface areas of the meat cubes are measured directly. For the sampling cloth treatment, the meat is arranged in a 10 cm by 10 cm block and the sampling cloth is applied to the top surface. Each mini-sampling cloth is extracted into 200 ml of phosphate-buffered saline (PBS). As a control, beef stew cubes are stomached for 60 seconds in 200 mL of PBS. All stomached cubes are measured to estimate surface area.The counts are normalized to the surface area and calculated as averages. This normalization allows for a comparison of similar situations.

[0072] This laboratory-scale experiment study was implemented and the average results are tabulated and shown in Table 1 of FIG. 3. These were analyzed with a General Linear Model (GLM) which indicates that contact time was not a factor. Multiple contacts produced slightly less than the expected increase and are truly additive.

[0073] A logical extrapolation of this exercise is to estimate the effective sample size of a sampling. This may not be truly possible given the differences in geometry. However, in one or more use cases when using only one side, a sampling cloth is about 6 times larger than those used in laboratory-scale experiments, and the intended use is to sample almost 907 kg (2000 lb) of product. Thus, it is reasonable to state that the effective sample is expected to be 136 to 181 kg (300 to 400 lb). Larger-scale experiments may be Petition 870240102506, dated 02 / 12 / 2024, pp. 30 / 95 / 49, implemented that may further confirm this estimate. In summary, a benefit and advantage of the above sampling method and apparatus may include providing an improvement over traditional random sampling for meat sampling by providing larger effective samples. SAMPLING EXAMPLES: LABORATORY-SCALE EXAMINATION OF SAMPLING EFFICIENCY FOR LETTUCE

[0074] Based on one or more cases, an example from a laboratory-scale study demonstrates a sampling efficiency of approximately 30% with respect to stomaching. It also shows that transfer is essentially instantaneous and that repeated contact collects more organisms. These observations confirm the intuitive assertion that continuous sampling will yield better information than random sampling for lettuce.

[0075] For example, according to a laboratory-scale experiment study, purchased lettuce is inoculated by immersion in a mixed culture of generic E. coli at ~105 CFU / ml. The E. coli is selected as benign and easy to enumerate. The lettuce is left to stand at 4°C for 30 minutes to allow adhesion. This short time may explain the higher efficiency observed when compared to a study with meat where adhesion may be faster. Then the six treatments listed in Table 2 shown in FIG. 4 can be performed in 5 replicates. All sampling cloths can be cut in half, 30 cm by 20 cm (12 in by 8 in), to reduce sample requirements and facilitate experiment execution. The sampling cloth can be, for example, a MicroTally cloth, but is not limited to this. The surface areas of the lettuce leaves can be measured directly.For sampling cloth treatments, the lettuce is arranged in a 10 cm by 10 cm block and the sampling cloth is applied to the top surface. Each mini-sampling cloth... Petition 870240102506, dated 02 / 12 / 2024, page 31 / 95 / 49 is extracted in 200 mL of PBS. As a control, lettuce can be stomached for 60 seconds in 200 mL of PBS. All stomached leaves are measured to estimate surface area. Counts are normalized to surface area and averaged. This normalization provides a comparison of similar situations.

[0076] This laboratory-scale experiment study was implemented and the average results are tabulated and shown in Table 2 of FIG. 4. These were analyzed with a GLM model which indicates that contact time was not a factor. Multiple contacts produced slightly less than the expected increase and are truly additive.

[0077] A logical extrapolation of this exercise is to estimate the effective sample size of a sampling. This may not be truly possible given the differences in geometry. However, in one or more use cases when using only one side, a sampling cloth is about 6 times larger than that used in the laboratory-scale experiment study, and the intended use is to sample almost 907 kilograms (2000 pounds) of product. Thus, it is reasonable to state that the effective sample is expected to be 180 to 272 kg (400 to 600 pounds). Larger-scale experiments can be implemented that may further confirm this estimate. In summary, a benefit and advantage of the above sampling method and apparatus may include providing an improvement over traditional random sampling for hardwood sampling by providing larger effective samples.

[0078] In one or more cases, swabs and / or plates, generally referred to as a plate below, may be used in accordance with one or more embodiments of this disclosure. A plate may include a microbial sampling material, such as sterile woven and / or nonwoven synthetic fabrics and nonwoven cloths for sampling and testing in the field of food safety. These plates may be folded and curved to Petition 870240102506, dated 02 / 12 / 2024, page 32 / 95 / 49 better conformity with the product streams (e.g., food) being sampled or when used in a manual mode as directed by the product and the container. In some cases, configurations for sampling raw products or materials may include a sampling device that moves through the stationary product effectively, producing the equivalent of a product stream when sampling needs to occur before harvest. In some cases, configurations may include tail-shaped plates similar, for example, to the tentacles of a jellyfish.

[0079] In one or more cases, configurations may be provided that may provide easier use under certain conditions. In one case, a microbial aggregating sampler may include a cover containing a microbial sampling material with a pocket formed in the cover to receive a limb or tool for handling the cover.

[0080] In some cases, the cover may also include a fastening feature formed in the pouch to receive the tool. The fastening feature may include at least one of a hole formed through the cover; a handle positioned inside the pouch to receive one end of the tool through it; or a tab positioned inside the pouch for one end of the tool to be secured to it.

[0081] In some cases, the cover may include a sheath formed in the pouch to receive a finger of a limb. In some cases, the pouch is formed through the cover such that the limb or the tool for handling the cover extends through the cover. The cover may be completely formed from the microbial sampling material. In some cases, the cover includes two plates fastened together to form the pouch. In some cases, the cover may include a single plate folded and fastened to itself to form the pouch.

[0082] For example, a sheet can go through bending and stitching to Petition 870240102506, dated 02 / 12 / 2024, page 33 / 95 / 49, to form a bag or pouch that can be used as a mitt, glove, sock, or other covering to facilitate manual sampling. Such a covering may enclose a limb, such as a hand, to facilitate pushing and pulling the sampler through the product to be sampled. In some cases, aggregating samplers may be created that have more hand-like configurations such as cuffs with thumbs and / or gloves with fingers, allowing the sampler to better conform to the hand. Such configurations may allow for easier use when the product is more viscous or more prone to adhering to the sampler. A benefit of this aggregating sampler with a pouch may include the sampler's ability to increase or maximize contact with the product when working with the sampler.Furthermore, in some cases, the addition of one or more fastening features, such as handles or tabs, to aid in controlling contact with the product may be included. These are representative examples and are not intended to limit other configuration options for an aggregating sampler.

[0083] In some cases, the aggregating sampler can be used in an automated machine setting to sample a product stream. The aggregating sampler may include various modifications depending on one or more cases. In some cases, the aggregating sampler may include one or more bends and curves that may improve usability and ease of use. In some cases, modifications may include, but are not limited to, forming a tube that can slide on one or more axes for positioning in the product flow. This configuration may eliminate the need to slide the sampler within any containment device. In some cases, the addition of tabs or holes may be provided that may allow positioning and facilitate attachment. In some cases, the active sampling surface may be attached to a support material or netting that has one or more of these features. One or more of these cases and modifications Petition 870240102506, dated 02 / 12 / 2024, pp. 34 / 95 / 49, may help facilitate contact with the product stream with minimal manual intervention.

[0084] FIG. 5 illustrates an aggregating sampler 500 which includes a pouch according to aspects of the present disclosure. As shown, the aggregating sampler 500 includes a cover 504 made of a microbial sampling material. The cover is formed such that it includes a pouch formed in the cover to receive a member or tool 502 inside the pouch for handling the cover.

[0085] FIG. 6 illustrates an aggregating sampler 600 that includes a pouch extending or being formed through a cover 604 according to aspects of the present disclosure. The pouch is formed such that the member or tool 602 for handling the cover 604 extends through the cover.

[0086] FIG. 7 illustrates an aggregating sampler 700 with at least one convex surface of the cover 704 according to aspects of the present disclosure. The cover forms a pocket for a tool or member 702. FIG. 8 illustrates an aggregating sampler 800 that includes at least one concave surface of the cover 804 according to aspects of the present disclosure. The cover 804 forms a pocket for a tool or member 802. FIG. 9 illustrates an aggregating sampler 900 that includes a curved cover 904 according to aspects of the present disclosure. The cover 904 is formed to include a pocket for a tool or member 902.

[0087] FIG. 10 illustrates an aggregating sampler 1000 which includes a hole or loop 1008 according to aspects of the present disclosure. The cover 1004 may include the hole or loop 1008, as positioned within the cover pouch 1004 and fixed to an inner surface of the cover 1004. This hole or loop 1008 may be used by a hook 1006 of a tool 1002 to attach to the cover 1004. Petition 870240102506, dated 02 / 12 / 2024, page 35 / 95 / 49

[0088] FIG. 11 illustrates an aggregating sampler 1100 that is formed as a mitten or glove for a hand 1102 according to aspects of the present disclosure. As shown, the cover 1104 of the aggregating sampler 1100 is formed to contour a hand 1102. In particular, a sheath is formed in the cover 1104 in this embodiment to receive a thumb of the hand 1102, although one or more sheaths may be used to receive any finger of the hand 1102. Furthermore, although only a hand 1102 is shown as an example member in this embodiment, the present disclosure is not thus limited, since other members (i.e., a foot) may be considered for other embodiments without diverging from the scope of the present disclosure.

[0089] In one or more cases, the aggregated sampling plate may be suspended within an active zone of a wash line with a rope or cable. This line may be attached in various ways to the sampling material, such as, for example, a metal eyelet and clamp. In some cases, a ball and sliding clamp may be used. In some cases, a drilled hole may be used, but it may be pulled out. In other cases, various other clamps may be used. In one or more cases, an optional float such as a fishing float with or without a weight may be included which will add drag and inflation that may improve surface exposure. This approach may not include a fixed device for holding the sampling material. Extraction Examples

[0090] The reference method for extracting microorganisms from random samples is homogenization by mixing or stomaching with an appropriate amount of fluid, usually a buffer. The purpose of adding fluid is to neutralize any antimicrobial or other sample properties that may be unfavorable for microbial growth, such as low pH and microorganisms suspended in the liquid, to facilitate the Petition 870240102506, dated 02 / 12 / 2024, page 36 / 95 / 49 downstream test. However, adding fluid also dilutes the concentration of microorganisms in the sample. Because most microbiological tests only take a portion of the homogenized material (e.g., 0.1 mL or 1 mL for plating; 2.5 g / L for direct PCR), homogenization by dilution decreases the detectability of organisms of interest by as much as 1,500,000 times. To increase detectability, a prolonged enrichment procedure is incorporated to allow a single viable cell to proliferate into millions so that it can be detected. This procedure takes 24 to 48 hours to obtain detection data and more days in decision-making time due to subsequent confirmatory steps. It is also important that the samples are properly preserved and that extraction is performed as close to the sampling time as possible.If the sample changed before extraction, the results do not represent the batch tested.

[0091] For the most conventional sampling programs, the percentage of negative samples exceeds 99%. Based on simple modeling, it can easily be concluded that the number of cells of the organism of interest is rarely greater than 1 and probably rarely greater than 5 based on a Poisson distribution. With so few cells present, enrichment is often done in the extraction buffer. This is critical with small sampling, but does not make this type of test more representative of the batch under test.

[0092] However, this conventional enrichment and detection can be applied to extract from an aggregating sampler when the presence or absence of the target organism is the desired goal. The detection modes are considered here.

[0093] These same considerations apply when extracting sample materials. However, the impact of failing to extract an organism is of an order of magnitude or less. Petition 870240102506, dated 02 / 12 / 2024, pp. 37 / 95 / 49 due to the larger effective sample size. Furthermore, the cassettes provide the opportunity to begin extraction more quickly by adding the fluid immediately after sampling, providing maximum time for extraction to occur while avoiding human interaction.

[0094] When extracting a 60x20 cm (24x8 inch) sample plate from a polyolefin sampler, a volume of 100 to 200 ml of extraction solution is generally appropriate. The composition of this solution is directed by the planned detection system and is usually defined in the associated method. EXAMPLES OF CONCENTRATION

[0095] During concentration, two classes of materials may need to be removed: small, molecular weight <100,000, and very large, >50 microns. Additionally, the sample may need to be concentrated to about 1 mL to be compatible with the screening detection system. Given that extraction usually starts at about 200 mL, there is a large amount of water to remove.

[0096] Two schemes are practical. Traditionally, one can filter the extraction fluid through an inert filter with a 50-micron cutoff and then pellet the organisms of interest by centrifugation. The resulting pellets can be resuspended in an appropriate buffer and taken for cleaning. Alternatively, after filtration, small molecules and water can be removed osmotically with adsorbents or pressure and a semipermeable membrane such as used for reverse osmosis or ultrafiltration. The latter is more accessible for automation since the resulting concentrated sample remains in solution. However, this option may require a purpose-built module to be implemented.

[0097] It is important to maintain the connection with the initial Meta data throughout this process. If enough interfering material is removed and the sample is sufficiently concentrated, the cleaning step examined at Petition 870240102506, dated 02 / 12 / 2024, page 38 / 95 / 49, can be skipped and proceed directly to the screening determination. This decision determination can be made on a case-by-case basis.

[0098] It is also possible to use non-specific linkage, such as a cation exchange surface, to collect target organisms and remove them from the main solution. Such an approach would partially combine concentration and scavenging. This approach is more practical when there are few larger particles to interfere with. EXAMPLES OF CLEANING

[0099] At this point in the process, the samples have been greatly reduced in volume, but the organisms of interest have not been segregated from other organisms, so the signal-to-noise ratio is still problematic. Furthermore, additional concentration may be required for detection without enrichment, during which one organism is converted into many at the cost of time and delays in decision-making.

[00100] Several schemes are practical, but all involve binding the organisms of interest in a small area such as a microfluidized or nanofluidized channel. This channel or area may or may not be filled with surface-activated nanofibers. Using elastoinertial microfluidics, viscoelastic flow enables migration based on the size of larger particles in a non-Newtonian solution, while smaller bacteria remain in the flowline of the blood sample inlet and can be separated. Surface-activated magnetic particles are being considered; however, the mechanical manipulation of these particles to obtain the desired small volume is a greater engineering challenge than activating the small surface area. Nevertheless, any binding geometry that fixes the organisms of interest and any other organisms selected to represent the other microflora in an appropriately small volume can be used. Petition 870240102506, dated 02 / 12 / 2024, p. 39 / 95 / 49

[00101] Surface activation may require numerous active binding sites in immediate proximity. SnapDNA mixed DNA primer arrays are one class of materials. Another class is an antibody cocktail for all organisms of interest.

[00102] The binding mechanism can bind all organisms of interest. These organisms can be sufficiently bound so that other organisms and materials are selectively removed from the binding area as clean fluid is passed through the channel.

[00103] The driving force to move the cells through the channel can be the mechanical action of the fluid flow, electrostatic since the surface of most bacteria is negatively charged, or a size-based pumping action such as that practiced with ferromagnetic particles. EXAMPLES OF SCREENING DETECTION

[00104] With the partially purified organism or organisms of interest clustered in a small area or small volume, if the cleaning step proves unnecessary, many approaches are available for screening detection for process control. The functional requirements are clearer. Firstly, enrichment culture consumes substantial amounts of time and this must be eliminated or reduced in one or more cases. Secondly, the screening metric may need to be a suitable index for statistical process control. This implies a measure with many states as opposed to the binary 0 and 1. The magnitude of this metric can refer to the degree of deviation from normal operation and therefore the probability that an outbreak may occur. Under these conditions, deviation detection can be based on classical rules for control mapping.Furthermore, trend detection rules have the potential to detect problems before significant deviations have occurred. Statistically grounded Westcard rules provide a basis for trend analysis. Petition 870240102506, dated 02 / 12 / 2024, pp. 40 / 95 / 49

[00105] Expressed differently, an index may need to record the relative presence of beneficial organisms and potential pathogens and aggregate this information in a useful way. As more information is acquired about specific products, the power of large-scale data will come into play. However, at the simplest levels, the aggregate level of potential pathogens is a useful screening tool. The relative balance between potential pathogens and beneficial organisms is a more sophisticated analysis to compensate for the seasonal variations that are inherent in many products. It is reasonable to expect to develop indices that are product-specific.

[00106] The information behind the indices is evolving rapidly. The simplest useful index is a ratio of pathogen intensity to a benign organism. These can be generated by many means including classical enumeration with plating, but classical methods are too slow to meet the outlined functional requirements. However, given the concentration of organisms in the cleaning substrate or concentrated extraction, it is possible to go directly to qPCR in some cases to generate index data.

[00107] There are two schemes for generating this type of data at its most sophisticated level. First, one can use a collection of ligands (antibodies, aptamers, or others) that bind and label all organisms of potential interest, producing a collection of signals that are multiplexed within a family of useful channels. Alternatively, one can generate an array of specific binding interactions that are analyzed chemometrically to produce a metric. The latter approach will be faster and likely less expensive after the search and analysis are done.

[00108] As an example of the first scheme, it would be to use a mixture of conjugated antibodies to bind to all types of cells. Petition 870240102506, dated 02 / 12 / 2024, p. 41 / 95 / 49 interest. For products, E. coli, Salmonella, and hemolytic Listeria are of enormous interest. Those for poultry focus on Campylobacter and Salmonella. Other industries have other and additional interests. These antibodies can be bound to organisms attached to the cleaning substrate. Labels retained with a fluorescent probe or an enzyme provide amplification and signal detection. When the cleaning region is as small as a cross-section, the signal from hundreds to thousands of cells on the cleaning substrate is detectable. Micro- and nanofluidization are required. However, specific detection protocols can be evaluated on a macro scale using an appropriately instrumented microscope that can be used to measure the signal from a small area where cells have been collected.To accelerate market entry, multiple detectors can be driven in parallel or series to generate similar multiple data channels.

[00109] The alternative scheme invokes the laboratory-on-a-chip concept. By constructing an array of binding sites, the composition of the samples can be consulted. PCR can selectively amplify the contents with a collection of primers. Such detectors may evolve to provide both screening detection at this stage and final secondary screening. However, it is likely to remain a two-step process due to economic considerations.

[00110] Several technologies can meet these requirements including, but not limited to, for example: 1) Sensors where the impedance of vibrational frequencies (which may, for example, include optical waveguides), or other properties of a transistor are modified by attaching the organisms of interest to the sensor. This approach may require the sensor to be built on the surface of the channel. Another technology may include, 2) qPCR where cells are deposited in place and the number of copies of the organisms of interest or the number of ribosomes of the Petition 870240102506, dated 02 / 12 / 2024, page 42 / 95 / 49 organisms of interest are estimated. Another technology may include, 3) The use of labeled antibodies to highlight the organisms of interest so that they can be detected on the absorbent surface spectrophotometrically. Enzymes, fluorescent probes and other materials to amplify the signal may be used.

[00111] It may be desirable to avoid confirming the presence of one or more specific pathogens during this screening. This is where the exchange between LOD and speed for useful information comes in. If the signal is not about three times the background, no further action is warranted.

[00112] Given that economics can play a role in testing, it should be mentioned that the sampling approach is compatible with various pooling strategies. Samples can be pooled anywhere along the processing path before detection. In various situations, one position will be more advantageous than others. Pooling samples before extraction reduces labor but reduces the resolution of results if a positive result is found. However, if the result is negative, tremendous savings are achieved. After concentration and cleaning, it would be possible to retest the sampled sample if only a portion of the extracted sample were used in a wet pooling approach, which can avoid detection costs. Each system needs to be evaluated to determine how best to control costs. EXAMPLES OF CONFIRMATION

[00113] The confirmation step will be applied when there is reason to suspect that a pathogen is present. Technology in this area is evolving rapidly with many new approaches and efficiencies being developed and introduced. Some will use the suggested techniques to screen with more specific reagents for confirmation. Currently, all common procedures rely on molecular biology or ligand binding reactions. Several strategies have been developed to amplify the Petition 870240102506, dated 02 / 12 / 2024, page 43 / 95 / 49 signal-to-noise ratio and to identify the contaminant for the desired specificity. The desired specificity varies from simple speciation to identifying specific serotypes.

[00114] For confirmation, a concentrated sample such as that provided in the screening detection above may be included. Concentration may be included due to the small volumes that are compatible with these types of procedures. Organisms, organism surface antigens, or organism nucleic acid may need to be extracted from the screening system to the extent that these materials can interact with the reagents of the confirmation procedure. In other words, there is no priority reason why the detection module should not be engineered for a second round of chemistry. Any of these materials may contain the information necessary to characterize the contaminant. Several such processes exist and are being improved. The choice of approach will be driven by cost, desired specificity, and desired speed.

[00115] Many strategies are available to amplify the baseline signal of these materials, which will still be present only in modest concentrations in the typical sample. The material yield from the hundreds to thousands of organisms of interest linked to the screening detector platform is still very small. Radioisotope-based amplification is largely obsolete, but still possible. However, enzyme systems are still in use, and new enzyme strategies are still being developed, such as those used for ELISA methods or with a luciferase. Fluorescent labels on specific antibodies, such as those proposed for screening determination, are less useful for this purpose due to the high potential for cross-reactivity. However, antibodies of this type are the basis for serotyping classifications, which until recently were the standard for characterization. These serotyping assays Petition 870240102506, dated 02 / 12 / 2024, p. 44 / 95 / 49 older individuals may require isolation and growth of organisms.

[00116] Increasingly, characterization is based on the presence of nucleic acid sequences. These can be nuclear DNA, ribosomal RNA, or messenger RNA. At the extreme, it is now common practice to sequence the entire genome of the organism. Increasing specificity is useful for identifying the source of a contamination. However, it also presents a potential probability in the case of a disease outbreak.

[00117] Molecular assays are built around the use of enzymes to replicate nucleic acid sequences to generate a signal large enough for detection. Various primers are used to select which portions are copied; up to and including the entire genome. For measurement, various fluorescent tags are used. One of the newest techniques utilizes the fusion and ligation of target material to known sequences to generate a complex array of information that can be used chemometrically instead of complete sequence data. Examples of Report and Scrolling

[00118] Both screening detection and confirmation results are reported directly to a sequence database. This report may not require human intervention if the included quality assurance standards (positive and negative controls) fall within normal ranges. This avoids transcription and transposition errors. Digital records are more reliable and accurate than manual records.

[00119] Both detectors should be part of the “Internet of Things”. This connectivity allows results to be delivered to operators on the floor, enabling rapid product release or product re-disposal if a potential problem is identified that may need to be addressed. Once the results are in an appropriate database, multiple users can have customized interfaces to provide the information. Some may Petition 870240102506, dated 02 / 12 / 2024, page 45 / 95 / 49, needs to track individual results. Others may be more interested in trends and averages. A class of users may be provided who may wish to aggregate even larger datasets to compare across locations.

[00120] There are many platforms available for extracting information from the database. For example, but not limited to these, Ignition has proven useful in this regard as it is open source allowing customization.

[00121] According to one aspect of the disclosure, a method for microbial sampling of food may include obtaining a microbial sample from one or more food items, extracting microorganisms from the microbial sample, concentrating the microorganisms, cleaning the microorganisms, recording a relative presence of microorganisms and any potential pathogens, aggregating information from a microorganism record from the microorganism record into a microorganism report, confirming the microorganism record, and reporting the microorganism report from the microorganism record.

[00122] In some cases, obtaining microbial sampling from one or more food items includes sampling, using an aggregating sampler, one or more food items that include a production batch of product or meat, creating one or more samples that comprise the microbial sampling. One or more samples may be configured to be processed to indicate whether pathogens are present in no more than a normal background.

[00123] In some cases, the method may additionally include evaluating, using an aggregating sampler, a level of cross-contamination control to validate or verify a washing process. Obtaining microbial sampling from one or more food items may include providing an aggregating sampler at a sampling location. The location Petition 870240102506, dated 02 / 12 / 2024, page 46 / 95 / 49 of sampling may be at least one among in a field, at harvest, immediately after unloading or cutting, in a washing system or after the washing system.

[00124] In some cases, extraction includes enriching the microbial sample and adding fluid to the microbial sample. In some cases, concentration includes filtering the extraction fluid from the microbial sample using at least one of centrifugation filtration or osmotically filtering. In some cases, cleaning includes binding microorganisms in a small area including one or more microfluidized or nanofluidized channels. In some cases, recording includes using a collection of ligands that bind and label all microorganisms of potential interest, producing a collection of signals that are multiplexed within a family of useful channels. In some cases, the ligands include one or more antibodies, primers, and aptamers.

[00125] In some cases, recording includes generating an array of specific binding interactions that are analyzed chemometrically to produce a metric. The method may additionally include constructing an array of binding sites, where the composition of the samples can be consulted and the contents selectively amplified using PCR with a collection of primers.

[00126] In some cases, confirmation includes extracting surface antigens from organisms or nucleic acid from organisms in a screening system to the extent that these materials can interact with the reagents of a confirmation procedure. In some cases, confirmation also includes additionally amplifying a baseline signal from the microorganism registry.

[00127] In some cases, the method may also include the use of an index as a substitute for direct results regarding the presence or absence of organisms of interest. In some cases, the method may also include the use of statistical process control to detect deviations in Petition 870240102506, dated 02 / 12 / 2024, page 47 / 95 / 49 microbial flora.

[00128] According to one aspect of the disclosure, a method of applying aggregation sampling to food items including providing at least one aggregation sampler at one or more sampling locations and sampling, using at least one aggregation sampler, a batch of produce or meat, creating one or more samples that comprise a microbial sampling.

[00129] In some cases, one or more samples are set up to be processed to indicate whether pathogens are present in no more than a normal background. In some cases, the one or more sampling locations include at least one in a field, at harvest, immediately after unloading or cutting, in a washing system, or after the washing system. The method may additionally include assessing, using the aggregating sampler, a level of cross-contamination control to validate or verify a washing process.

[00130] According to one aspect of the disclosure, an apparatus for microbial sampling, including means for obtaining a microbial sample from one or more food items, means for extracting microorganisms from the microbial sample and means for concentrating the microorganisms, means for cleaning the microorganisms, means for recording a relative presence of microorganisms and any potential pathogens, means for aggregating microorganism record information into a microorganism report, means for confirming the microorganism record and means for reporting the microorganism report of the microorganism record.

[00131] According to one aspect of the disclosure, an apparatus for microbial sampling includes at least one processor configured to generate control signals to control obtaining a microbial sample from one or more food items, extracting microorganisms from the microbial sample, concentrating the microorganisms, cleaning the Petition 870240102506, dated 02 / 12 / 2024, page 48 / 95 / 49 microorganisms, recording a relative presence of microorganisms and any potential pathogens, aggregating microorganism record information into a microorganism report and confirming the microorganism record and a transmitter configured to transmit the microorganism report from the microorganism record. In some cases, the device may additionally include an aggregating sampler configured to obtain microbial sampling.

[00132] According to one aspect of the disclosure, a non-transient, computer-readable means for microbial sampling having instructions stored therein for obtaining a microbial sample from one or more food items, extracting microorganisms from the microbial sample, concentrating the microorganisms, cleaning the microorganisms, recording a relative presence of microorganisms and any potential pathogens, aggregating information from the microorganism record into a microorganism report, confirming the microorganism record and reporting the microorganism report from the microorganism record.

[00133] According to one aspect of the disclosure, a method for sampling food including concentrating microorganisms and removing interference, recording a relative presence of microorganisms and any potential pathogens, and aggregating microorganism record information into a microorganism report. According to one aspect of the disclosure, a system capable of implementing one or more of the new aspects discussed in this application disclosure.

[00134] According to one aspect of the disclosure, a microbial aggregating sampler, including a cover including a microbial sampling material with a pouch formed in the cover to receive a limb or tool for handling the cover.

[00135] In some cases, the cover includes a fastening feature formed in the pouch to receive the tool. In some cases, the feature of Petition 870240102506, dated 02 / 12 / 2024, p. 49 / 95 / 49, the fastening includes a hole formed through the cover, a handle positioned inside the bag to receive one end of the tool through it, and a tab positioned inside the bag for one end of the tool to be fastened to it.

[00136] In some cases the cover includes a sheath formed in the pouch to receive a finger of a limb. In some cases the pouch is formed through the cover such that the limb or tool for handling the cover extends through the cover. In some cases the cover is completely formed from the microbial sampling material. In some cases the cover includes two plates fastened together to form the pouch. In some cases the cover includes a single plate folded and fastened to itself to form the pouch. OTHER APPLICATIONS BESIDES BACTERIAL TESTING

[00137] The aggregating sampler can be used to sample additional analytes besides bacteria, including yeast, molds, viruses, allergens, toxins such as aflatoxin, or particulates such as dust. Commonality is the presence on the surface of the analyte at low levels that can be concentrated. The basic process is the same with the same key steps. The biggest differences will be in the detection strategy, but these strategies are well known to those who study these analytes.

[00138] The methods disclosed herein comprise one or more steps or actions to obtain the described method. The steps and / or actions of the method can be interchanged with each other without departing from the scope of the claims. In other words, unless a specific order of steps or actions is specified, the order and / or use of specific steps and / or actions can be modified without departing from the scope of the claims.

[00139] As used herein, a phrase referring to “at least one of” a list of items refers to any combination of those items, including unique members. As an example, “at least one of: a, b, or c” is Petition 870240102506, dated 02 / 12 / 2024, p. 50 / 95 / 49 intended to encompass a, b, c, ab, ac, bc, and abc, as well as any combination with multiples of the same element (for example, aa, aaa, a-ab, aac, abb, acc, bb, bbb, bbc, cc, and ccc, or any other order of a, b, c). As used herein, including in the claims, the term “and / or,” when used in a list of two or more items, means that any of the listed items may be employed alone or any combination of two or more of the listed items may be employed. For example, if a composition is described as containing components A, B, and / or C, the composition may contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[00140] As used herein, the term “determination” encompasses a wide variety of actions. For example, “determining” can include calculating, computing, processing, deriving, investigating, searching (e.g., searching in a table, a database, or another data structure), ascertaining, and the like. Also, “determining” can include receiving (e.g., receiving information), accessing (e.g., accessing data in memory), and the like. Furthermore, “determining” can include resolving, selecting, choosing, establishing, and the like.

[00141] The preceding description is provided to enable anyone skilled in the art to practice the various aspects described herein. Various modifications to these aspects will be readily apparent to those skilled in the art, and the generic principles set forth herein may be applied to other aspects. Thus, the claims are not intended to be limited to the aspects shown herein, but should be given the full scope consistent with the language of the claims, wherein reference to an element in the singular is not intended to mean “one and only one” unless specifically so stated, but rather “one or more”. For example, items “one” and “an” as used in this application and in Petition 870240102506, dated 02 / 12 / 2024, p. 51 / 95 / 49 attached claims should generally be interpreted to mean “one or more” unless otherwise specified or clear from the context to be directed to a singular form. Unless specifically stated otherwise, the term “any” refers to one or more. Furthermore, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or”. That is, unless otherwise specified or clear from the context, the sentence, for example, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, for example, the sentence “X employs A or B” is satisfied by any of the following cases: X employs A; X employs B; or X employs both A and B.All structural and functional equivalents for the elements of the various aspects described throughout this disclosure that are known or may later become known to those of common skill in the art are expressly incorporated herein by reference and are intended to be covered by the claims. Furthermore, nothing disclosed herein is intended to be dedicated to the independent public unless such disclosure is explicitly cited in the claims. No element of a claim shall be construed under the conditions of 35 USC §112, sixth paragraph, unless the element is expressly cited using the phrase “means to” or, in the case of a method claim, the element is cited using the phrase “step to”.

[00142] The various method operations described above may be performed by any suitable means capable of performing the corresponding functions. The means may include various hardware and / or software components and / or modules, including, but not limited to, a circuit, an application-specific integrated circuit (ASIC), or a processor. Generally, where operations illustrated in the figures exist, these operations may have corresponding counterpart means-plus-function components with similar numbering.

[00143] The various illustrative blocks, modules and logic circuits Petition 870240102506, dated 02 / 12 / 2024, page 52 / 95 / 49 described in connection with this disclosure may be implemented or realized with a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device (PLD), discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general-purpose processor may be a microprocessor, but alternatively, the processor may be any commercially available processor, controller, microcontroller, or state machine.A processor can also be implemented as a combination of computing devices, for example, a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[00144] If implemented in hardware, a hardware configuration may comprise a processing system in a wireless node. The processing system may be implemented with a bus architecture. The bus may include any number of interconnecting buses or bridges depending on the specific application of the processing system and the overall design constraints. The bus may link together various circuits including a processor, machine-readable media, and a bus interface. The bus interface may be used to connect a network adapter, among other things, to the processing system via the bus. The network adapter may be used to implement the signal processing functions of the PHY layer. In the case of a user terminal 120 (see FIG. 1); a user interface (e.g., keyboard, screen, mouse, joystick, etc.) may also be connected to the bus.The bus can also connect various other circuits such as timers, peripherals, voltage regulators, etc. Petition 870240102506, dated 02 / 12 / 2024, page 53 / 95 / 49 power control circuits and the like, which are well known in the art and therefore will not be described. The processor may be implemented with one or more general-purpose and / or special-purpose processors. Examples include microprocessors, microcontrollers, DSP processors, and other circuits that can execute software. Those skilled in the art will recognize how best to implement the described functionality for the processing system depending on the particular application and the overall design constraints imposed on the overall system.

[00145] If implemented in software, functions can be stored or transmitted as one or more instructions or code on a computer-readable medium. Software should be broadly interpreted to mean instructions, data, or any combination thereof, whether referred to as software, firmware, middleware, microcode, hardware description language, or otherwise. Computer-readable media include both computer storage media and communication media, including any means that facilitate the transfer of a computer program from one place to another. The processor may be responsible for controlling the bus and general processing, including the execution of software modules stored on computer-readable storage media. A computer-readable storage medium may be coupled to a processor such that the processor can read information from the storage medium and write information to it.Alternatively, the storage medium may be integral to the processor. For example, machine-readable media may include a transmission line, a data-modulated carrier wave, and / or a computer-readable storage medium with instructions stored on it separate from the wireless node, all of which can be accessed by the processor via the bus interface. Alternatively or in addition, machine-readable media or any... Petition 870240102506, dated 02 / 12 / 2024, page 54 / 95 / 49. A portion of these may be integrated within the processor, as may be the case with cache and / or general register files. Examples of machine-readable storage media may include, by way of example, RAM (Random Access Memory), flash memory, phase-change memory, ROM (Read-Only Memory), PROM (Programmable Read-Only Memory), EPROM (Erasable Programmable Read-Only Memory), EEPROM (Electronically Erasable Programmable Read-Only Memory), registers, magnetic discs, optical disks, hard disks, or any other suitable storage media or any combination thereof. Machine-readable media may be embodied in a computer-program product.

[00146] A software module can comprise a single instruction or many instructions and can be distributed across several different code segments, between different programs, and across multiple storage media. Computer-readable media can comprise multiple software modules. Software modules include instructions that, when executed by a device such as a processor, cause the processing system to perform various functions. Software modules can include a transmit module and a receive module. Each software module can reside on a single storage device or be distributed across multiple storage devices. For example, a software module can be loaded into RAM from a hard drive when a triggering event occurs. During the execution of the software module, the processor can load some of the instructions into the cache to increase access speed.One or more cache lines can then be loaded into a general register file for execution by the processor. When referring to the functionality of a software module below, it will be understood that such functionality is implemented by the processor when executing its instructions. Petition 870240102506, dated 02 / 12 / 2024, page 55 / 95 / 49 software module.

[00147] Also, any connection is properly termed a computer-readable medium. For example, if software is transmitted from a website, server, or other remote source using a coaxial cable, fiber optic cable, twisted pair, digital subscriber line (DSL), or wireless technologies such as infrared (IR), radio, and microwave, then coaxial cable, fiber optic cable, twisted pair, DSL, or wireless technologies such as infrared, radio, and microwave are included in the definition of medium. Disc, as used herein, includes compact disc (CD), laser disc, optical disc, digital versatile disc (DVD), floppy disk, and Blu-ray® disc where discs usually reproduce data magnetically, while discs reproduce data optically with lasers. Thus, in some respects, computer-readable media may comprise non-transient computer-readable media (e.g., tangible media).Furthermore, for other aspects, computer-readable media may include transient computer-readable media (e.g., a sign). Combinations of the above should also be included within the scope of computer-readable media.

[00148] Thus, certain aspects may comprise a computer program product for performing the operations presented herein. For example, such a computer program product may comprise a computer-readable medium having instructions stored (and / or encoded) therein, the instructions being executable by one or more processors to perform the operations described herein. For example, the instructions for performing the operations described herein and illustrated in the attached figures.

[00149] Furthermore, it should be evaluated whether the modules and / or other appropriate means to execute the methods and techniques described herein can be downloaded and / or otherwise obtained by a user terminal and / or base station as applicable. For example, such a device could be coupled to a Petition 870240102506, dated 02 / 12 / 2024, page 56 / 95 / 49 server to facilitate the transfer of means to perform the methods described herein. Alternatively, several methods described herein may be provided by means of storage media (e.g., RAM, ROM, a physical storage medium such as a compact disc (CD) or floppy disk, etc.), such that a user terminal and / or base station may obtain the various methods upon coupling or provide the storage media for the device. In addition, any other suitable technique for providing the methods and techniques described herein to a device may be used.

[00150] It should be understood that the claims are not limited to the exact configuration and components illustrated above. Various modifications, changes, and variations may be made to the arrangement, operation, and details of the methods and apparatus described above without departing from the scope of the claims. Petition 870240102506, dated 02 / 12 / 2024, page 57 / 95

Claims

1 / 5 CLAIMS 1. A method for microbial sampling of food, characterized in that it comprises: obtaining a microbial sample from one or more food items; extracting microorganisms from the microbial sample, wherein the microorganisms include non-pathogenic microorganisms and any potential pathogens, if present; concentrating the microorganisms; cleaning the microorganisms; determining a quantity of microorganisms and determining the presence of any potential pathogens; aggregating information from the determination of the quantity of microorganisms into a microorganism report; confirming the determination of the quantity of microorganisms; and reporting the microorganism report of the determination of the quantity of microorganisms and the presence of any potential pathogens.

2. Method according to claim 1, characterized in that obtaining microbial sampling of one or more food items comprises: sampling, using an aggregating sampler, multiple food items from a production batch of product or meat, creating one or more aggregated samples that make up the microbial sampling, wherein the aggregating sampler is a contact sampler comprising a microbial sampling material having an external sampling surface for contact with food items to be sampled.

3. Method according to claim 2, characterized by the fact that the aggregating sampler comprises: a cover comprising a microbial sampling material with a pocket formed in the cover to receive a limb or tool for handling the cover.

4. Method according to claim 3, characterized in that the cover comprises a fastening feature formed in the pocket to receive the tool and wherein the fastening feature comprises one of: a hole formed through the cover; a handle positioned inside the pocket to receive one end of the tool through it; and a tongue positioned inside the pocket for one end of the tool to be fastened to it.

5. Method according to claim 3, characterized in that the pouch is formed through the cover such that the limb or tool for handling the cover extends through the cover.

6. Method according to claim 3, characterized in that the cover comprises: two sheets fastened together to form the pouch or a single sheet bent and fastened to itself to form the pouch.

7. Method according to claim 1, characterized in that it further comprises: evaluating, using an aggregating sampler, a level of cross-contamination control to validate or verify a washing process.

8. Method according to claim 1, characterized in that obtaining microbial sampling of one or more food items comprises: providing an aggregating sampler at a sampling location, Petition 870240102506, dated 02 / 12 / 2024, page 59 / 95 3 / 5 wherein the sampling location is at least one of: in a field, in a combine harvester, at harvest, immediately after unloading or cutting, in a washing system or after the washing system.

9. Method according to claim 1, characterized in that the extraction comprises: enriching the microbial sample; and adding fluid to the microbial sample.

10. Method according to claim 1, characterized in that the concentration comprises: filtering the extraction fluid from the microbial sample using at least one of centrifugation filtration or osmotic filtration.

11. Method according to claim 1, characterized in that the cleaning comprises: binding microorganisms in a small area including one or more microfluidized or nanofluidized channels.

12. Method according to claim 1, characterized in that determining the quantity of microorganisms comprises: using a collection of ligands that bind and label all microorganisms of potential interest, producing a collection of signals that are multiplexed within a family of useful channels, wherein the ligands include one or more antibodies, primers and aptamers.

13. Method according to claim 1, characterized in that determining the quantity of microorganisms comprises: generating an array of specific binding interactions that are analyzed chemometrically to produce a metric comprising: constructing an array of binding sites, in which a composition of the samples can be consulted; and amplifying, using PCR, the samples selectively with a collection of primers. Petition 870240102506, dated 02 / 12 / 2024, p. 60 / 95 4 / 5 14. Method according to claim 1, characterized in that the confirmation comprises: extracting surface antigens from microorganisms or nucleic acid from microorganisms from a screening system to a degree that these materials can interact with reagents of a confirmation procedure; and amplifying a baseline signal in determining the quantity of microorganisms.

15. Method according to claim 1, characterized in that it additionally includes the use of one or more indices as a surrogate to direct results regarding the presence or absence of organisms of interest or a statistical process control to detect deviations in the microbial flora.

16. A method for applying aggregation sampling to food items, characterized in that it comprises: providing at least one aggregation sampler in one or more sampling locations, wherein the aggregation sampler comprises a microbial sampling material having an external sampling surface for contacting the items to be sampled and having a pocket for receiving a tool or appendage or having an apparatus for holding and positioning the microbial sampling material during sampling; and sampling, using the at least one aggregation sampler, multiple food items from a production batch of product or meat, creating, by contacting the various food items with the aggregation sampler, one or more aggregate samples that comprise a microbial sample.

17. Method according to claim 16, characterized in that it further comprises: Petition 870240102506, dated 02 / 12 / 2024, page 61 / 95 5 / 5 determining a quantity of microorganisms in one or more aggregated samples; indicating whether pathogens are present in no more than a normal background based on the determination of the quantity of microorganisms.

18. Method according to claim 16, characterized in that one or more sampling locations include at least one in a field, at harvest, immediately after unloading or cutting, in a washing system or after the washing system.

19. Method according to claim 16, characterized in that it further comprises: evaluating, using the aggregating sampler, a level of cross-contamination control to validate or verify a washing process.

20. Method according to claim 17, characterized in that the quantity of microorganisms is determined from one or more aggregated samples without enrichment. Petition 870240102506, dated 02 / 12 / 2024, p. 62 / 95

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