TR2021018888A2 - A HERBAL EXTRACT MIX AND METHOD OF OBTAINING ANTIMIROBIAL EFFECT ON WATER LIFE - Google Patents

Abstract

The invention generally relates to a therapeutic and growth-promoting aquatic living, antimicrobial effective herbal extract mixture containing Artemissia absinthium essential oil, Artemissia absinthium extract, vegetable oil and emulsifier elements, and the method steps that enable this mixture to be obtained.

Description

DESCRIPTION A WIFE OF VEGETABLE OZW WITH ANI'iiiiiiKRoeiYAL EFFECT ON WATER LIFE AND METHOD OF OBTAINING TECHNICAL FIELD Meet. antimicrobial effect for the aquaculture industry. protozoa group in farm fish therapeutic and growth promoting herbal medicine with antiparasitic effect against parasites Concise is about a mixture and the etherne method of obtaining it.

KNOWN STATE OF THE TECHNIQUE The healthy alternative herbal products used today in aquaculture although the benefits have been sought for a long time. especially for such products Studies on its use in the field of aquaculture are very limited. Animal Bioactive containing herbal substances applied against diseases seen in production It was determined that very good results were obtained against injective agents thanks to the substances. (Harikrishnan et al. 2010).

Today, medicinal plants and herbal medicine raw materials. in the treatment of water products forms a part of the drugs used. like synthetic metronidazole As a result of the emergence of cytotoxic and mutagenic harmful effects of some chemicals restricting or prohibiting their use. infectious parasitic natural to be used in combating factors. reliable and economical new products increased the necessity of using it. Therefore, in the field of aquaculture production (in aquaculture) antiparasitic treatment of medicinal plants to combat fish pathogens Various studies have been conducted on its effects (Chitmanat et al. 2004: Moon et al. 2012). Puk et al. (2014).

Use of herbal products; easier to obtain, relatively inexpensive. have minimal side effects, are generally effective at low doses, and broad spectrum (bacterial, viral, fungal, parasitic) against pathogens. has increased the possibilities of being preferred today.

Lots of scientific research done. gram (+) and gram (-) bacteria of herbal products It shows strong antibacterial effect on strains and is used as a natural antibiotic. demonstrated that they can be used safely.

While there is only one bioactive substance in the composition of synthetic antibiotics. There are many bioactive substances in natural herbal products. herbal products It is also preferred that their biological recycling does not harm the environment. are among the reasons.

Nitroimidazole group drugs and metronidazole treatment of bacterial and parasitic diseases used in veterinary medicine. However, due to the carcinogenic properties of this drug, therapeutic use in animal production. Number 613I98 in European Union countries prohibited by the directive. Intestinal Hexamita enlekslyonlan (Hexamltlosis.) is used for trout in aquaculture production. It is a protozoan parasitic disease that causes serious mortality in farms.

This disease, which has an epizootic character, was diagnosed in the Black Sea region of our country in 2002. While there was a mortality of up to 32% in trout farms located in It caused 20% mortality in the Marmara region. In the fish farms of the disease it is possible to see it especially from December to August (Ogut and Akyol. 2005; Timur et al., 2009). The breeding period of the parasite is rapid in the acute phase of infection. and while it's short. chronic lon's extensive period between spring and autumn. This causes mortality to occur throughout the year (Govind. 2013). is becoming. The difference between the intensity of the infection and its prevalence in this disease. relationship is not well known. food by damaging the intestinal epithelium of the parasite It is considered to be in competition with substances. Infections are in the gut it is chronic. While quarantine practices are beneficial for Hexamita infections, Good breeding and nutritional practice are important in conservation. parasite of your disease It has been reported that cysts and trophozoites are transmitted by oral ingestion. fish population its density is effective in the formation of the disease (Woo. 1999) Nitroimidazole by feed in the treatment of parasitic Hexamita infection dlmetrldazol and metronldazol from the group are applied. With this The carcinogenic effect of metronidazole from the nithioimidazole group and its DNA and cell structure Its use in the European Union countries due to the damage on the with the prohibition of alternative treatments and products that are safe for humans and the environment. revealed the need for further investigation.

One of the inventions in the literature, TR 2015 14573 Invention with class A23K 20110IPC. used in the textile industry. 'Growth and “Development-promoting and immune-enhancing organic feed additive” The invention, in its 'special part, is to reduce production costs in fish farming. to prevent losses due to diseases and to increase productivity; your fish organic feed additive that promotes growth and development and strengthens immunity as substance. devenger apparatus with water distillation method from orange peel Essential oil will be obtained by using The essential oil obtained is used in fish feeds. the immune system will be strengthened-' contains information- One of the inventions in the literature. TR 2001 01074 A231( Invention with 1/16 IPC class. Titled "Antimicrobial enzymes in animal feed" meet. in the summary 'Poultry, pigs. dairy calves and fish for growth and improvement of feed conversion rate. monogastric or ruminant Use of two antimicrobial enzymes for use in feed for non-animal animals has been explained. This practice helps the farmer use growth promoting antibiotics. able to gain its reputation. Enzymes have antibacterial activity and while the enzyme can break down the cell membrane of bacteria (eg lysozyme). another can produce a compound (eg, oxidase) that is toxic to bacteria. These enzymes It results in a polyunsaturated fatty acid (PUFA) such as arachidonic acid. is being increased.' contains information.

One of the inventions in the literature. TR 2005 02655 numbered Invention with class A011( 61/00 iPC, 'rainbow trout growing' Ion balancing in the rations (feeds) used." effect on performance in rainbow trout. Control for research .2% By adding CaCl2. 2% Salt. 2% NaZCOS, 2% NaHCOB. 4% NAZCOS and 4%NaHCO3 Rainbow trout with different CAD are used. rainbow aiabatigi fattening With the increase of the CAD value ( in their feeds, the new feed is cationic. The growth increased in rainbow trout and feed conversion coefficient has decreased, while the CAD degen is increased, that is, the feed is made cationic. the sources of cation additives used. Na2CO3 and NaHCOS, which also increase the Na/K ratio It has been seen that it is." When the application is examined, the feeds used for their fish are among the mentioned feeds. mentioned, but it is clear that these feeds do not have a therapeutic effect.

As a result, it is a therapeutic and growth-promoting antimicrobial in aquatic organisms. an effective herbal "ozfu mixture" and improvements in the method of obtaining it. going. therefore, it will eliminate the disadvantages mentioned above and There is a need for new structures that will bring solutions to existing systems.

OBJECTIVE OF THE INVENTION The present invention satisfies the above-mentioned requirements. all the disadvantages therapeutic and therapeutic in aquatic organisms, which eliminates A mixture of herbal extracts with antimicrobial effect that promotes growth and obtaining it relates to method.

The main purpose of the invention. is to obtain a completely natural and herbal product mixture.

Since it is a completely natural and herbal product, it does not have any side effects.

Another purpose of the invention. It is therapeutic against parasites. In the process of treating herbal products, by influencing the biochemical processes of microbial cells It damages the physicochemical integrity. Herbal products, broad spectrum antimicrobial are families. They show similar activity against parasites and bacteria. Especially hydrophobic. terpenes in the structure. cell wall of the microorganism by interacting with the cell wall It damages the cell wall integrity by disrupting the structure of the proteins that hold the wall together. is suffering. The hydrophobicity of terpenes is associated with the lipids in the cell wall. interaction causes lipids to aggregate together and increase membrane permeability. causes. Naturally, the deterioration of the physicochemical structure causes protons in the cell. motion and electron flow, and hence transport, and the cell causes coagulation of its contents. Microorganism with impaired cell permeability Depending on the concentration of H+ and K+ galleons in the cytoplasm, water losing. causes the death of parasites.

For this purpose, terpenoids were used in chemical analyzes of Artemisia species used in this invention. and flavonide components were determined to contain terpenoid and It has antimicrobial effects originating from sesquiterpene Iactone compounds (Diler et al. Terzioglu. 2014). Terpenoids are the most studied metabolites in the genus Artemisia. A It is reported that there are 4 major compounds in the absinthium species. These are p-thuyon. pee- epoxyocimene trans-sahinylacetate and chrysantenly are common (ngen et al. 2004. Bora and Sharma.2011) naturally infected with the parasite Hexamita salmonis in Caismamia. Ethanol extracts of Artemlsia abslnthium L. plants in juvenile rainbow trout calculated. The number of parasites in herbal extract groups from the 11th day has been found to decrease.

Another purpose of the invention is to increase growth in aquatic organisms. In recent years, medical In order to increase the growth performance of essential oils obtained from plants, It has come to the fore to be added to feeds (Hammer et al. 1999). indirectly in fish Feed additives that increase growth regulate the intestinal microbiota, intestinal modoloisin has a positive effect. Appetite in animals and fish in animal production increases. Also alkaline phosphatase. peptidase. maltase gut like sucraz and Iactaz Increases the activation of enzymes, by regulating the balance of intestinal microflora increases the growth performance of fish as a result of increasing nutrient absorption. it leads. If the intestinal microflora of fish is not properly controlled negatively affects host resistance (Zheng et al. 2009). That's why. herbal products increase the number of beneficial bacteria. Therefore, the harmful fiora the number of bacteria is reduced. Therefore, the control of mlcrothoranin with herbal products It can positively affect the performance of fish and increase growth positively.

Artemisia species have been used as medicinal plants since ancient times. Antiparasitic. with anti-inflammatory, anti-microbial and antirheumatic properties plant Artemisia species phytochemically; essential oils. terpenes. phenolic aromatic It is an important group of medicinal plants (Kordaii) because it has aliphatic and aliphatic components. et al.. 2005; Abad et al. 2012). wormwood bitter wormwood. known by local names such as white wormwood Artemisia absinthium L. This plant, which can grow up to 120 cm, is grayish or whitish. It is green in color, with segmented leaves. Widely distributed in Anatolia and naturally growing is a perennial plant. It usually grows in the Mediterranean region. It has red flowers.

A. absinthium has anthelmintic properties (Baytop. 1999).

Artemisia group plant species and Artemisia absinthium plant species with antiparasitic activity. is known. For example, Huang et al. (2013). Against Dactylogyms intermedius parasite Inoelemisier and Caesalpinia anthelmintic effect of some herbal extracts. lysima Dryopteris crassimizoma against the intermedius parasite. kochia polygala They examined the effect of tenuifolia plants and gave 22-97 mgll to Dryopteris plant. It has been determined that it is 100% effective after 48 hours at the concentration. Ekanem and Brisibe (2010). Arlemisia annua extract fry Heterobranchus longifilis fish It has been used for treatment against monogenean parasite in the genus species. In this study to juvenile fish at concentrations of 50 and 200 mgIL in the bapa in soil ponds. application has been made. This is where parasites in the gills are counted with a stereomicroscope. In this study, artemisinin, which is an extract of A. annua, parasitized in H. long'philis fish species. It is stated that it is effective against.

Oriakpono et al. (2012). Extract of Artemisia annua Saratherodon melanotheron fish investigated the effect of the species against the monogenean parasite. For this purpose 5. 10. 20, 40. Concentrations of 80 mgIL were also selected and 2 mglL were parasiquental for comparison. used. Terpenoids of A. annua. flavonoids. comerins and essential oils It was determined that the plant extract, which was prepared at a dose of 80 mgi'L, was determined to contain determined that it reduced the number of parasites from 60 to 12, for which it was the most effective dose. a. Toxic effect on fish in treatment applications made from annua extract not seen.

Antiparasitic effect of Artemisia species on human and animal parasites There is also some research on the subject. omega. Tariq et al. (2009), made in their research. wormwood (Artemisia absinthium) extract in vivo in sheep They investigated the effects of clay worms on their eggs in feces under these conditions.

Ethanol extract was found to be as effective as albendezole and 2 g kg-1 (body weight) is 90.46% in the number of eggs in the stool in 15 days. When 1 g kg-1 is given They saw an 80.49% decrease in the census. Dose of wormwood when considering clay worms its effect is significant. In this study, wormwood extract was found in sheep. they stated that it can be an alternative to lowering drugs.

Petinotus has traditionally been used as a parasitic in skin animals and humans. is being used. Valderrabano et al. (2010). in four-month-old lambs in their study. They added 20% wormwood (Artemisia absinthium) straw to ground pellet alfalfa. and evaluated its effect on clay worms. As a result, Haemonhcus oontortus (clay worm) fecal egg excretion 73% on a dry matter basis It is determined that it takes place.

Bora and Sharma (2010). Petinotu (Artemisia absinthium) in their study When 1.6 9 kg-1 of the extract was given to rabbits, no toxic effect was observed. ethanol and aqueous extract showed antiheimintic effect against intestinal worms in sheep.

Trypanosoma brucei Trypanosoma cruzi. Leishmania infantum and Plasmodium faloiparum; Antifungal effect against Microsporum canis and Candida albicans 300 mg kg-1 methanoi extract against Triohinella spiraiis in rats. that you are effective. 'artemisetin' isolated from wormwood against melanoma B 16 showed antitumor activity. that it delays the development of tumor tissue. continually It has been reported that its use has a neurotoxic effect due to thujone and its derivatives. they did.

Caner et al. (2008). In their study, Trichineila spiraiis parasite in mice methanoi extract of wormwood (Artemisia absinthium) 300 per body weight When given mg kg-1, it reduces the rate of larvae in the tongue by 635%. capsule in the larval stage found that when 600 mg kg-1 was given, the rate of larvae decreased by 599%.

Brisibe et al. (2008), in their study, another type of wormwood, sweet Adding wormwood (A. annua) leaves to chicken and layer hen rations 10% and 20% Fattening performance and egg criteria were examined. A. annua in chickens When the leaves are given, the development of parasites is prevented. feed consumption and live that the weight increases. significant difference in the number of varieties in the stool samples compared to the control group. They found that the degree of decrease. Parra et al. (2001). pelinctu in their work in vivo and in vitro studies in mice to determine the lethal dose of eketraktin. They determined that the dose was 3.19-238.55 pg mL-1. As a result, Artemisia Potential of ethanol or methanol extract of the plant for parasite treatment Due to the carcinogenic effect of existing drugs As an alternative Artemisia absinthium is a plant-derived product against fish parasites in aquaculture for the first time. used as a reliable new product for the environment and human health. As a result of the results, filing a patent application came to the agenda.

Table 1. Medical effects of some Artemisia species spreading in the world (Abad et al. 2012) Type Location Oulllk Ariemieia abaintiiium to Turkish Sib'I European Antharaeitic Artemisia campestris Turkey Amtiarasilik Artemisia abysslnlca Saudi Arabia Antibacterial Artemisla afra South Africa Diabetes traditional treatment Artemisia annua Asia, Europe, North America Malatya traditional cure Artemisia arberesicens Mediterranean region Anti-inflammatory Artemisla argyl China. Japan. Russia Herbal Pharmaceuticals Artemieie biennis North America Antlhelmlm Artemieia drecunculue Russia. France Herbal medicine Artemisia echegarayi Argentina Feed additive Artemisia lukuda Korea. Japan. Taiwan Antlbaderial Artemisia harbe-alba Jordan Calmstiria'. fire & driver Artemisia princepe Japan Traditional treatment of circulatory diseases Arlemisia mbripes Korean Stomachache. hemostatitis agent Artemlsla scoparla Central European Anthakterlyel. Ineektlsld. openness Artemisia spicigera Middle East Anatolian Leather. for ulcer discomfort Artemis-ie tridenlata North America Feed source (animals) Members of the Hexamitidae family of the order Diplomonadida. high oxygen tolerance flagellates and l-lexamita, Octomitus. It consists of the genera Giardia and Spironucleus.

Hexamita spp. Duýardin (1838) members, living in anaerobic sediments. mammal. vomit reptil. They are animals that can settle in the digestive tract of amphibians and fish. hexamita spp. warm water. It settles in the gastrointestinal tract of coldwater and aquarium fish.

Angelfish and discus species of aquarium fish are due to this parasite. serious health problems are seen. Stress conditions cause rapid growth of the protozoan parasite. may cause proliferation and the emergence of clinical symptoms. in fish The species causing the disease are H. salmonis, H. truttae, H- intestinalis. Floyd and Reed (1994). Weak and stressed fish are more susceptible to heavy infestation. of hexamitiosis symptoms; weight loss. decrease in activity. not feed. infected angel fish swimming horizontally and sagging abdomen. the rank of the infections of the intestinal limen preparationin 200)( and 400:: light done by microscopic examination. The parasite attracts attention with its fast swimming movements. Floyd and Reed (1994). Through bait in the treatment of parasitic Hexam'rta infection dimetridazole and metronidazole are administered. However, metronidazole DNA and It is used in aquaculture because of its harmful effects on cell structure. Its use in the European Union countries is prohibited by the directive numbered 6121/98. Recurrence possibilities of hexamitiosis inductions and used in their treatment The necessity of chemicals and drugs to be safe for humans and the environment is an alternative in treatment. revealed the necessity of investigating the components.

The herbal product has the potential to be produced by culture and is found in nature and It is a product that is not difficult to obtain. For this reason, culture in the production of aquatic organisms It can be easily applied and given by adding to the bait of fish in fishing. features.

Making the most of its advantages with the structuring of the present invention and additional elements To be understood, it should be evaluated together with the figures explained below. DETAILED DESCRIPTION OF THE INVENTION In this detailed explanation. The subject of the invention is therapeutic and growth-enhancing aquatic organisms. A herbal extract with antimicrobial effect that supports it and the method of obtaining it preferred configurations are only for a better understanding of the subject and It is disclosed that no limiting effect will occur. Invention in general. on the basis of. Artemisia absinthiurri essential oil. Artemisia In aquatic organisms containing absinthium extract, vegetable oil and emi'ilgalibrden elements therapeutic and growth promoting. A blend of herbal extracts with antimicrobial effect and this is about the method steps that make the mixture happen.

In the table given below, the usable percentage amounts by weight of the said properties and the preferred e_diien Wide amount is given Aspect Name By Weight Preference Can Be Used By Weight Amount (%i) Amount (%) Artemlsia absinthium 1 0.51 essential oil Artemisla absinthium 1 0.54 extract Vegetable oil 97 98.1- 96 The essential oil of Artemisia absinthium characterizes the invention. Artemisia It consists of absinthium extract, vegetable oil and em'i'ilgat'or.

In addition, 0.5 - 1% by weight Artemisia is the characteristic feature of the invention. absinthium essential oil, 0.5 - 1% Artemisla absinthium extract, 0.9 - 2% Materials with emi'ilgatbr ratio, vegetable oil ratios of 98.1% - 96% forms.

Properties of Raw Materials in Buiug: Artemieia absinthium essential oils; myrcene (8.6-22.7%). dihydrochamazulene ieomer (5.5-11.6%), germacrene D (2.4-8.0%). ß-thujone 7.0) components. Terpenic substances that make up the majority (90%) monoterpene It exists in the form of sesclterpene and diterpene. terpenes Oxygenated derivatives formed by oxidation. unique to essential oils the smell of it. It gives its taste and therapeutic properties. 2. Artemlsia abslnthlum extract 58 different components in the chemical composition of Artemisia absinthium extract was obtained from A. absinthium. rich in monoterpene compounds ( has not been determined. Because Terpenes are hydrophobic, they are lipids in the cell wall. With its interaction with the lipids, it is possible to collect lipids together and to increase the permeability of the membrane. causing an increase. Disruption of phycochemical structure naturally. Proton movement and electron flux and hence transport in the cell causes disruptions and coagulation of cell contents (Erdogan et al. Everest 2013) Chemical of Artemisia absinthium L. plant extract sabinol (42.22%), one of the monolerpenes in its composition. chrysanthenylacetate (14.73%). epoxy-ocimene (497%). monoterpene thujone (219%), d-lsothuione (397%). B selinene (260%), a sesquiterpene. alkyl hexanal (230%), which is an aldehyde. cymene (171%), which is a monoterpene, and benzaldehyde (1% .04). 3. Vegetable oil As a vegetable oil. commercial sunflower oil can be used. Sunflower (Helianthus annuus L.) pressing from the seeds of the plant. extraction etc. from transactions clear, obtained by refining after passing. liquid it is rain. Purpose of usage. Artemisia absinthium essential oil and Artemisia absinthium in order to facilitate the mixing of the extract with the food. is used. 4' E ..i i.. In this invention, polysorbate 80 can be used as emulsifier. How to use the emulator purpose Artemisla absinthlum extract. Artemisia absinthium essential oil and herbal It is used to homogenize the components of the oil.

Complex containing essential oils and extracts of medicinal plants, different bioactive components Since they are mixtures, they are stronger if used together in terms of their biological effects. show effect. Their mechanism of action depends on the active ingredients and their properties. it is attached. Artemisia absinthium essential oil and extract; terpenes, phenolic It has antimicrobial properties because it is available in aromatic and aliphatic compounds.

Terpenic substances that make up the majority of essential oils (90%) It exists in the form of monoterpene, sesquiterpene and diterpene. terpenes Oxygen derivatives formed by oxidation. unique to essential oils the smell of it. It gives its taste and therapeutic properties. Artemisia absinthium extract Its antiparasitic effect has been determined in various studies. the plant in question The extract is commercially available for the treatment of nematode infections in sheep. It has been reported that it can be used (Tarlqa et al., 2009). Effective against parasites of plants The extract form is used to make it. In general, to obtain an extract; dried herbal material is used.

A solvent such as liquid ethanol is used to obtain the extract. herbal in this It takes 5 to 8 days for the solvent to act completely in the material. This At the end of the period, the extraction solvent is removed and the resulting extracts are collected. Then to turn this extract into a clear solution. centrifugation and filtering process is done. Essential oils are bactericidal. virucidal. in fungicidal, antiparasitic, insecticidal, medical and cosmetic applications. especially the last in pharmacology over the years. extensively in agriculture and the food industry This volatile aromatic structure, which is mostly obtained by distillation, is used. oil components, volatile molecules such as terpenes, tierpenoids, phenol derivatives a complex structure consisting of compounds and allyl'attic substances contains. With cytoplasm coagulation caused by essential oils, Iipit and The damage of proteins is concentrated and as a result, in the cell wall and Damage to the membrane causes lysis by affecting the flow of macromolecules. is happening. In this study, to obtain an effective result against parasites, oil and extract mixture was used together. Production Process; Preparation of Plant Samples To obtain essential oils from plant samples; Samples were dried in the shade. After separating from the stems, the flowers and leaves are blended into powder. has been brought. After the samples are passed through a 2 mm sieve, they are kept in the room until they are used. It was stored in glass jars at warm temperatures (Kirbag and Zengin. 2005; Güllüce Obtaining Essential Oil by Distillation Method 200 g of herbal powder for hydrodistillation process done in Clevenger apparatus After each of the material is filled into a 5 liter balloon, 2 lt of water is added and (Distillation by adding 100 ml of water to 10 g sample) jacketed heater It was boiled for 4 hours with the help of (Pirbalouti et al. 2009b; Pirbalouti et al. 2010a).

The finger mounted on the water vapor and accompanying oil balloon The steam that is dragged into the cooler in the form of tap water and hits the cooler. It was deposited in the separation cabinet as drops by condensation (Figure). Intensity In order to remove the oil remaining on the top in the separation cabinet due to the difference, the water in the lower part has been removed. The oil is then filled with sodium sulfate (moisture in the oil). to be kept) in the ependorls found and stored at 48°C until used. (Toroglu and Cenet, 2006; Baydar, 2009). In that application known methods are used.

Preparation of Ethanol Extract of Artami'si'a absi'nthium L. Plants g dry plant sample was kept in 50 ml of 70% ethanol for 2 hours and then filtered. For the process of whatrnan N01 filter paper was passed. with the help of a water bath The evaporation process was done and the solution was used at -18°C until it was used. is hidden.

Fish of Artemisia absinthr'um L. Essential Oil and Artemisia absr'nthi'um Extract Adding to Yams 100 ml product to commercial fish food (Artemisia absinthlum essential oil, Artemisla absinthium extract. emulsifier and vegetable oil mixture) to 1000 9 fish feed It is added by the spray method. weekly in order to maintain the effectiveness of the components. it is prepared. +-t't:i is also stored in glass bottles with lids.

Claims (1)

REQUESTS . The invention is a herbal extract with antimicrobial effect, therapeutic and supporting growth in aquatic creatures. Artemisia absinthium essential oil. Artemisia absinthium extract. vegetable oil and emi'iigatbri'i ipen'ne. . It is related to the herbal mixture suitable for Process 1, its feature is; 0.5 - 1% by weight Artemisia absinthium essential oil. 0.5 - 1% Artemisia absintiiium extract. 0.9 - 2% emi'ilgat'or. It is 98.1 - 96% vegetable oil content. . It is related to the herbal mixture according to claim 1. feature: sunflower oil is used as vegetable oil. Meet. therapeutic and growth promoting in aquatic organisms. It is a method of obtaining a herbal extract mixture with antimicrobial effect. feature; - Artemisia absinthium essential oil, Artemisia absinthium extract. em'uigatbr and includes the process step.