Botulinum Toxin Production Research Articles

Editorial Comment on: Recommendations on the Use of Botulinum Toxin in the Treatment of Lower Urinary Tract Disorders and Pelvic Floor Dysfunctions: A European Consensus Report

The authors are to be congratulated on this endeavour, which for the first time has provided an evidence-based literature review using the European Association of Urology (EAU) levels of evidence [1]. The review covers all of the current potential applications of botulinum toxin therapy attributed to lower urinary tract and pelvic floor dysfunction. Inevitably, one of the problems is that much of the current literature is based on small case series and there is little in the way of adequately powered controlled studies, although such studies are currently being conducted. A caveat that needs to be borne in mind when considering use of any type of botulinum toxin product is that different preparations are not interchangeable. This caveat refers not only to dosing recommendations but also to efficacy and safety data of a particular brand. To date, the majority of data published have used the botulinum toxin A preparation from Allergan. The vast majority of published data are derived from experience in the adult population and not the paediatric population; hence, extrapolation between different age groups cannot be carried out safely until further data are available. Based on the evidence available, the authors are able to provide a grade A level of recommendation for botulinum toxin type A for the treatment of the intractable symptoms of neurogenic detrusor overactivity (NDO) or idiopathic detrusor overactivity (IDO). They recommend caution in the context of IDO. We should recognise that, at present, this therapy is clearly effective for NDO and IDO and is being used on a named patient basis, and it is important that this is emphasised to patients. Regulatory trials are currently under way, and it will be interesting to see the outcome of those. There are certainly a number of other aspects which need to be investigated in the future, including the most effective dose in different clinical conditions and the site and depth of injection. Clearly, we also need longer term information relating to the consequence of chronic use of the therapy, both in terms of ultrastructural changes in the bladder and neural innervation.

Biosimilars: Illustration of scientific issues in two examples

Scientific issues and clinical implications associated with the use of biosimilars (biopharmaceuticals that are similar to an innovator product, notwithstanding minor differences) are illustrated in two examples, botulinum neurotoxins and erythropoietic agents. Comparison of Botox and Dysport, products that both contain botulinum toxin type A, revealed distinct differences in physicochemical characteristics, approved indications, dosing, and frequency of adverse events. Differentiating between botulinum toxin products on the basis of immunogenicity in the clinical setting would be of value in product selection, and pharmacists could play a valuable role in collecting antigenicity rate data and reporting them to the Food and Drug Administration (FDA) and the manufacturer. Various ethical and practical considerations are associated with the use of erythropoietic agents. The desire to optimize patient care and outcomes must be weighed against the likelihood of obtaining reimbursement for erythropoietic therapy, and reimbursement policies vary from one state to another. Comparing erythropoietic agents requires the use of a consistent and valid definition of treatment response. The definition of response that FDA will accept in the future when evaluating applications for approval of new biosimilar erythropoietic agents and establishing equivalence remains to be determined. A variety of scientific and practical clinical issues are associated with the use of biosimilars, including product differences in physicochemical characteristics, reimbursement policies, and the need for valid and clinically relevant criteria for comparing the efficacy and safety of biosimilars and innovator products.

Type C bovine botulism outbreak due to carcass contaminated non-acidified silage

The first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay. Clostridium botulinum type C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group III C. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibit C. botulinum toxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Safety of Botulinum toxin a in cerebral palsy

The use of Botulinum toxin (BoNT) in the treatment of focal spasticity in adults and children has become well established worldwide. Since the first use of BoNT in children with spastic equinus over 10 years ago, there have been no serious or unexpected safety concerns for this indication. However, within the last year, there has been media attention as well as reviews by European and US regulatory agencies about the safety of all marketed Botulinum toxin products for the way in which they are used currently. Following the pioneering studies by Koman in the US and Graham in Europe, the initial publications recommended utilizing doses of 1–4 U BOTOX/kg body weight (BW) per muscle in the gastrocnemius and soleus and a total dose of not more than 8 units BOTOX/kg BW. For Dysport, the recommended dosing was up to 30 U/kg BW up to a maximum of 1000 U. In a consensus statement published in 2000, Graham and colleagues recommended a maximum dose of 50 U of the BOTOX formulation in any one site, a maximum total dose of 12 U/kg BW or 400 units at an injection session. This was based on clinical experience current at that time. Recognizing the multilevel nature of spasticity in children with spastic diplegia, firstly Graham and colleagues, followed by Molenaers and colleagues, extended existing protocols to expand the muscle groups injected to include the hamstrings, adductors and iliopsoas. Although the dose per muscle was not altered, this resulted in a stepwise increase in total dose administered. Subsequently, there have been numerous publications on outcomes and safety of BOTOX when used in a multi-level approach. The most common, obvious treated related side effects are pain at the site of injection, excess weakness of the targeted muscle, and weakness of adjacent muscle. The 2000 consensus statement anticipated that the risk of adverse events in children with cerebral palsy could be related to the type of cerebral palsy than the dose of toxin administered. Specifically, it is anticipated that children at GMFCS level V, who are non-ambulatory and often debilitated with impaired attention and cognition; have comorbidities of epilepsy and other cardiac, pulmonary or neurologic conditions; and have symptoms of pseudo-bulbar palsy, might be specifically at risk for complications of BoNT and/or the procedure including the use of sedative agents and anaesthesia. In a recent randomized trial, Graham and colleagues carefully studied adverse events in a large number of children with cerebral palsy treated with an average dose of 14 U BOTOX/kg BW. There was evidence of possible systemic spread and adverse events in some children, but no fatalities directly related to the administration of the toxin. This experience is consistent with the literature. In a recent review of our BoNT database, we have identified the following risk factors for adverse events in the administration of BoNT. In order of importance these are: (1) the child's GMFCS level; (2) the total dose administered; (3) method of sedation/anaesthesia. Secondary factors for further investigation include: (1) current and past medical history, specifically dysphagia, aspiration, cardiopulmonary events, concomitant medications; (2) reconstitution volume; (3) total number of muscles injected; (4) single or divided dose per muscle; (5) method of muscle targeting, e.g. muscle stimulation versus ultrasound.

Comparison of type a botulinum toxin products in clinical use

Comparison of type a botulinum toxin products in clinical use

Tonsils – Place of botulinum toxin production: Results of routine laboratory diagnosis in farm animals

Since a case of a veterinarian was reported, who was likely to be infected/intoxicated by Clostridium botulinum during the handling of a diseased animal, tonsils in animals were tested for botulinum neurotoxin and bacterial forms of neurotoxic Clostridium botulinum during routine botulism laboratory examinations including standard samples (intestinal tract and liver) from 48 cattle, 11 horses, and 14 goats. Ten out of 60 samples from tonsils contained free botulinum toxin, and 12 out of 59 were positive for live toxin producing bacteria. In 32 out of 162 intestinal samples toxin was detected. Toxin producing bacteria were found in 37 samples. Eight of 56 liver samples contained free toxin, and 15 out of 43 toxigenic bacteria. Samples from 10 slaughter pigs were all negative, whereas from slaughter cattle tonsils had a high incidence of toxin (7 of 10) or toxigenic bacteria (2 of 8). The results are discussed in the context of effects on animal health and botulism as zoonosis.

Potency of the botulinum toxin product CNBTX-A significantly exceeds labeled units in standard potency test

Potency of the botulinum toxin product CNBTX-A significantly exceeds labeled units in standard potency test

Evaluation of the Effect of Acetylsalicylic Acid on Clostridium botulinum Growth and Toxin Production

The Republic of Georgia (ROG) has the highest incidence of botulism among all countries in the world, with most cases attributed to home-preserved vegetables. Based on epidemiologic data, the occurrence of botulism in ROG is lower in areas where aspirin (active ingredient, acetylsalicylic acid [ASA]) is added to home-canned vegetables. The objective of this study was to evaluate, with a broth medium, the antibotulinal activity of ASA to determine the possible role of ASA in preventing botulinum toxin production in home-canned vegetables. Trypticase-peptone-glucose-yeast (TPGY) broth (pH 7.0) with 0, 0.3, and 0.6 mg of ASA per ml was inoculated with a 10-strain mixture of proteolytic Clostridium botulinum type A and B spores at ca. 103 spores per ml. The inoculated broths were incubated at 31°C under anaerobic conditions, and C. botulinum growth and botulinum toxin production were determined for up to 36 h. Results showed ASA in broth delayed (time to initial detectable toxin produced and amount of toxin produced), but did not prevent, both growth and toxin production by C. botulinum. These results would not provide a definitive explanation for differences in toxin production in canned vegetables prepared with and without aspirin.

Botulism in 4 Adults Following Cosmetic Injections With an Unlicensed, Highly Concentrated Botulinum Preparation

Botulism is a potentially lethal paralytic disease caused primarily by toxins of the anaerobic, spore-forming bacterium Clostridium botulinum. Although botulinum toxin A is available by prescription for cosmetic and therapeutic use, no cases of botulism with detectable serum toxin have previously been attributed to cosmetic or therapeutic botulinum toxin injections. On November 27, 2004, 4 suspected botulism case-patients with a link to cosmetic botulinum toxin injections were reported to the Centers for Disease Control and Prevention. To investigate the clinical, epidemiological, and laboratory aspects of 4 suspected cases of iatrogenic botulism. Case series on 4 botulism case-patients. Clinical characteristics of the 4 case-patients, epidemiological associations, and mouse bioassay neutralization test results from case-patient specimens and a toxin sample. Clinical characteristics of the 4 case-patients were consistent with those of naturally occurring botulism. All case-patients had been injected with a highly concentrated, unlicensed preparation of botulinum toxin A and may have received doses 2857 times the estimated human lethal dose by injection. Pretreatment serum toxin levels in 3 of the 4 case-patients were equivalent to 21 to 43 times the estimated human lethal dose; pretreatment serum from the fourth epidemiologically linked case-patient was not available. A 100-microg vial of toxin taken from the same manufacturer's lot as toxin administered to the case-patients contained a toxin amount sufficient to kill approximately 14,286 adults by injection if disseminated evenly. These laboratory-confirmed cases of botulism demonstrate that clinical use of unlicensed botulinum toxin A can result in severe, life-threatening illness. Further education and regulation are needed to prevent the inappropriate marketing, sale, and clinical use of unlicensed botulinum toxin products.

Hazard and control of group II (non-proteolytic) Clostridium botulinum in modern food processing

Group II (non-proteolytic) Clostridium botulinum poses a safety hazard in modern food processing, which consists of mild pasteurization treatments, anaerobic packaging, extended shelf lives and chilled storage. The high risk is reflected in the relatively large number of botulism cases due to group II C. botulinum in commercially produced foods during the past decades. Because of the high prevalence of group II C. botulinum in the environment, food raw materials may carry spores. Although group II spores are less heat-resistant than group I (proteolytic) spores, they can tolerate the heat treatments employed in the chilled food industry. Some food components may actually provide spores with protection from heat. Spore heat resistance should therefore be investigated for each food in order to determine the efficiency of industrial heat treatments. Group II strains are psychrotrophic and thus they are able to grow at refrigeration temperatures. Anaerobic packages and extended shelf lives provide C. botulinum with favourable conditions for growth and toxin formation. As the use of salt and other preservatives in these foods is limited, microbiological safety relies mainly on refrigerated storage. This sets great challenges on the production of chilled packaged foods. To ensure the safety of these foods, more than one factor should safeguard against botulinal growth and toxin production.

Distribution of Dungeness Crab Meat to Retail Stores and Restaurants in Hermetically Sealed Five Pound Cans and the Risks of Clostridium botulinum Toxin Production

ABSTRACT Ready-to-eat picked meat of Dungeness crab (Cancer magister) packaged under vacuum in hermetically sealed (No. 10, five pound) cans, has become a public health concern because the product does not receive a process to inactivate Clostridium botulinum and refrigeration is the only barrier to prevent this bacterium from growing and producing neurotoxins. However, crab meat packaged in this manner has been distributed on ice to retail stores and restaurants for over 50 years without any reported illnesses from C. botulinum. Studies were therefore made to determine conditions that may have contributed to the safety of this product. C. botulinum type E was shown to be present in live Dungeness crab entering the processing facility, but was not detectable after the crabs were cooked in boiling water for ten minutes, nor in picked crab meat. Crab meat samples were inoculated with 10, 100, 1000 and 10,000 type E or type A spores/50 g, packaged in oxygen-permeable or vacuum packaged in oxygen-impermeable bags and stored at either 12°C or 25°C to determine whether spoilage occurred before the production of neurotoxins. Crab meat stored at 12°C in oxygen-impermeable bags were spoiled after 3 days and remained nontoxic for 20 days even when inoculated with 10,000 type E spores. Crab meat was spoiled after 24 hr at a gross abuse temperature of 25°C. Samples with type E spores became toxic in oxygen-impermeable bags between 6 and 13 days and only when inoculated with 10,000 spores. All samples with type A spores were nontoxic and very spoiled in oxygen-impermeable and permeable bags after 3 days at 25°C. Type A toxin was produced between 3 and 6 days and only in samples with 1000 and 10,000 spores. The inactivation of C. botulinum type E during the cooking step and rapid spoilage has undoubtedly played an important role in the safety of crab meat. However, to assure the safety from C. botulinum, No. 10 cans of crab meat must be stored and distributed on ice or at refrigeration temperatures below 3°C.

Pharmacology of Botulinum Neurotoxin Serotype A

An historical perspective on the discovery and development of botulinum neurotoxin for commercial use, the differences between various botulinum toxin serotypes and commercially available products, and the structure, mechanism of action, pharmacologic effects, and immunogenicity of botulinum toxin type A are discussed. Botulinum toxin was discovered several centuries ago, but its medical uses have only recently been explored. There are several botulinum toxin serotypes that differ in composition and molecular weight. The protein component of the macromolecular structure that has pharmacologic activity requires enzymatic activation, and the required enzyme varies by serotype. All serotypes interfere with muscle contraction by inhibiting acetylcholine release into the synaptic space at the neuromuscular junction, although the intracellular target varies by serotype. An antinociceptive effect may be mediated by a similar mechanism involving inhibition of pain mediator release. The effects of botulinum toxin usually are only temporary because collateral axonal sprouts that can release acetylcholine develop. The protein load and immunogenicity vary among the commercially available botulinum toxin products. The smallest protein load and longest dosing interval should be used to minimize resistance. Alternating among different serotypes is not an effective strategy for preventing or overcoming resistance because cross-resistance can occur. All botulinum toxin serotypes and products cause temporary muscle paralysis, yet vary in important ways that may affect their clinical use.

Could blackbird mortality from avicide DRC-1339 contribute to avian botulism outbreaks in North Dakota?

Blackbird (family Icteridae) depredation on sunflower (Helianthus annuus) crops in the prairie states of the United States has motivated the proposed use of an avicide, DRC-1339 (3-chloro-4-methylaniline), to decrease their numbers. The resulting mortality of black- birds at wetland roosts could increase the potential of avian botulism occurring in affect- ed marshes. To assess this possibility, we seeded (artificially placed) blackbird carcasses in selected wetlands in Stutsman County, North Dakota, during August-September 2000 and July-September 2001 to evaluate their rate of decomposition and role in initiating avian botulism outbreaks. We monitored carcasses to determine their persistence, the frequen- cy and amount of maggots produced, and the presence of type C botulinum toxin. In 10 of our 12 study wetlands, blackbird carcasses were not rapidly removed by scavengers, thus providing substrate for maggot growth and potential production of Clostridium botu- linum toxin. Decomposition of carcasses occurred rapidly, and maggot production aver- aged 4-5 g per carcass within 9 days. We were unable to detect C. botulinum type C toxin in any of the 377 blackbird carcasses or the 112 samples of maggots we collected in 2000 or 2001. None of the 25 blackbird carcasses we tested contained botulinum spores, the most probable explanation for the absence of botulinum toxin production. Our results indicate that the likelihood of DRC-1339-poisoned blackbirds causing botulism outbreaks would be minimal in North Dakota wetlands during late summer and early autumn.

Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids

The effect of fat on the antibotulinal activity of 11 food preservatives, 12 free fatty acids, and nine lots of enzyme-modified cheese (EMC) was evaluated in a media system. Anhydrous milkfat or soybean oil was added to tubes of Trypticase–peptone–glucose–yeast extract medium (TPGY) supplemented with the additives (final pH adjusted to 5.9). Treatments were inoculated with 3-log 10 proteolytic Clostridium botulinum spores/ml (10-strain mixture of serotypes A and B) and incubated anaerobically at 30°C for up to 14 days. For the preservative and fatty acids studies, growth of C. botulinum was determined by measuring optical changes at OD 640 nm . Botulinal toxin production was determined in EMC-treatments using the mouse bioassay. Data revealed that the antibotulinal effects of nisin, and free fatty acids caprylic, capric, lauric, myristic, oleic, and linoleic acids were each significantly reduced in treatments supplemented with 20% fat ( P<0.05). Similar trends were observed in TPGY supplemented with 20% fat and potassium sorbate, sorbic acid, monolaurin, polyphosphate emulsifier, or EDTA–lysozyme, but the differences were reduced. Fat was also antagonistic to the antibotulinal activity of five EMC-treatments. This study suggests that fat may reduce the efficacy of some antimicrobials added to or found naturally in foods.

Factors That Contribute to the Botulinal Safety of Reduced-Fat and Fat-Free Process Cheese Products

The effects of fat, type of natural cheese, and adjunct process cheese ingredients were evaluated to determine factors that contribute to the botulinal safety of reduced-fat (RF) process cheese products stored at 30°C. In the first set of experiments, pasteurized process cheese products (PPCPs) were formulated using full-fat (FF) Cheddar, 30% RF Cheddar, or skim milk (SM) cheese as cheese-base types and were standardized to 59% moisture, pH 5.75, 2.8 or 3.2% total salts, and 15 to 19% fat. Subsequent trials evaluated the effect of fat levels and adjunct ingredients in PPCPs made with SM, RF, and FF cheese (final fat levels, less than 1, 13, and 24%, respectively). When fat levels of PPCPs were comparable (15.1, 19.1, and 16.2 for product manufactured with SC, RF, and FF cheese, respectively), botulinal toxin production was delayed for up to 2 days in PPCPs formulated with SM compared with RF or FF cheese; however, the effect was not statistically significant. When fat levels were reduced to less than 1% in SM PPCPs, toxin production was delayed 2 weeks in products made with SM compared with RF or FF cheese manufactured with 13 or 24% fat, respectively. The antibotulinal effect of adjunct ingredients varied among the products manufactured with different fat levels. Sodium lactate significantly delayed toxin production (P < 0.05) for all fat levels tested, whereas β-glucan fat replacer did not delay toxin production. An enzyme-modified cheese used as a flavor enhancer significantly delayed toxin production (P < 0.05) in SM (less than 1% fat) products but had little to no inhibitory effect in RF (13% fat) and FF (24% fat) cheese products. Similarly, monolaurin increased the time to detectable toxin in SM products but was ineffective in RF or FF cheese products. These results verify that RF PPCPs exhibit greater safety than FF products and that safety may be enhanced by using certain adjunct ingredients as antimicrobials.

Antibotulinal Activity of Process Cheese Ingredients

Ingredients used in the manufacture of reduced-fat process cheese products were screened for their ability to inhibit growth of Clostridium botulinum serotypes A and B in media. Reinforced clostridial medium (RCM) supplemented with 0,0.5, 1, 2, 3, 5, or 10% (wt/vol) of various ingredients, including a carbohydrate-based fat replacer, an enzyme-modified cheese (EMC) derived from a Blue cheese, sweet whey, modified whey protein, or whey protein concentrate, did not inhibit botulinal growth and toxin production when stored at 30°C for 1 week. In contrast, RCM supplemented with 10% soy-based flavor enhancer, 10% Parmesan EMC, or 5 or 10% Cheddar EMC inhibited botulinal toxin production in media for at least 6 weeks of storage at 30°C. Subsequent trials revealed that the antibotulinal effect varied significantly among 13 lots of EMC and that the antimicrobial effect was not correlated with the pH or water activity of the EMC.

Co-incubation of Clostridium botulinum with protective cultures

Potential neurotoxin production by psychrotrophic Clostridium botulinum is the significant food safety risk in refrigerated foods of extended durability. Protective cultures (PC), nisin-producing Lactococcus lactis (6.3 × 10 5 cfu/ml) and pediocin A-producing Pediococcus pentosaceus (10 7 cfu/ml), were co-incubated with C. botulinum type B (6.2 × 10 2 cfu/ml) in tryptose, peptone, glucose and yeast extract broth for 31 days at 5 °C, 14 days at 10 °C and for 10 days at 15 °C. Bacterial populations, pH, botulinal toxin and bacteriocin production were measured. At 5 °C, the populations of all species did not change during incubation. At 10 and 15 °C, the PCs increased by 1–3log. At 15 °C, C. botulinum increased by 1.5–3log and was not inhibited by the PCs. At 10 °C, this pathogen was not detected after 10 days of co-incubation. The reduction in C. botulinum population coincided with the nisin level produced by L. lactis exceeding 100 IU/ml, but it was not rapid enough to prevent botulinal toxin formation.

Honey consumption in the state of São Paulo: a risk to human health?

Infantile botulism was recognized in 1976 as a paralyzing disease caused by the ingestion of viable spores that would germinate and colonize the intestinal tract of infants, with local production and absorption of Clostridium botulinum toxin. The possible origins of botulinic spores are dust and honey, which has been identified as a dietary risk factor for infantile botulism. The objectives of the present study were to investigate 100 honey samples obtained in the state of São Paulo (Brazil) in terms of incidence of botulinic spores and of microbiologic quality, in agreement with Decree 367/9. All 100 samples analysed were negative for the presence of Salmonella, Shigella, total coliforms. C. botulinum spores were present in 3 samples (3%) and molds and yeasts, in 64 samples (64%), but only 25 (25%) exceeded established criteria, with counts ranging from zero to 1.5×10 5 CFU/g. The presence of small sporogenic Gram-positive rods was observed in 42 (42%) of the 100 samples tested but these bacteria were not identified.

Survival of Clostridium botulinum in modified atmosphere packaged fresh whole North American ginseng roots

Fresh North American ginseng roots (Panax quinquefolius) were packaged in medium transmission film in air. Six roots were used per pouch. Anaerobic conditions developed during 2, 10 and 21°C storage, most rapidly at the elevated temperatures. Clostridium botulinum challenge tests were performed for 10 and 21°C samples. At 21°C storage, neither uninoculated control nor inoculated roots showed C. botulinum toxin production after 6 weeks of storage. At 10°C, C. botulinum toxin production occurred after 14 weeks in inoculated roots, while roots maintained an acceptable appearance. This highlights the potential risk of food poisoning by ginseng packaged under modified atmosphere when anaerobic conditions develop.

BOTULINAL TOXIN PRODUCTION IN VACUUM AND CARBON DIOXIDE PACKAGED MEAT DURING CHILLED STORAGE AT 2 AND 4C

This study was undertaken to determine if carbon dioxide packaging of meat afforded a food safety advantage over vacuum packaging with respect to botulinal toxin production during chilled storage. A cocktail of washed spores from five toxigenic clostridial strains – four reference Clostridium botulinumstrains [types A, B (2 strains) and E] and a C. butyricum type E strain – was inoculated onto lamb chumps. Of these strains, two were psychrotolerant. The inoculated chumps were individually carbon dioxide packaged and duplicate packs were placed into storage at 10, 8, 6, 4 and 2C. All storage regimens included a weekly defrost cycle when meat surface temperatures increased by up to 6 to 7C during a 2 to 2.5 h period. After 84 days storage, packs were assessed for the presence of botulinal toxin using the mouse bioassay procedure. All packs contained botulinal toxin. To compare toxin production in vacuum and carbon dioxide packs at chill temperatures, the challenge trials were repeated for 4 and 2C storage. Packs were examined at regular intervals for toxin presence. Both pack types contained toxin after 21 and 48 days storage at 4 and 2C, respectively. In the unlikely, but not impossible, event that raw meat would be contaminated with psychrotolerant toxincapable clostridial spores, product safety, with respect to botulinal toxin presence after prolonged chilled storage, requires storage temperatures to be maintained below 2C for both vacuum and carbon dioxide packaged product.