Plant to Extract ratios do not completely describe botanical extracts because other important factors influence the make-up of final extracts, such as the quality of the raw starting material (as can defined by pharmacopeial standards), extraction solvent(s) used, duration and temperature of extraction, and percentage and type of excipients present. Other important qualitative descriptions may include constituent &#;fingerprinting.&#; Despite these issues, Plant to Extract ratios are often used as a measure of extract strength for dosage calculations. This article defines and clarifies the meaning of Plant to Extract ratios and their proper use in describing and labeling botanical extract ingredients and finished products containing them.
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Dietary supplement current good manufacturing practice (cGMP) requires establishment of quality parameters for each component used in the manufacture of a dietary supplement to ensure that specifications for the identity, purity, strength, composition, and limits on contaminants are met. * Compliance with botanical extract ingredient specifications is assured by using scientifically valid methods of analysis, the results of which are reported on certificates of analysis (CoAs). However, CoAs routinely include additional data that are not amenable to verification through methods of analysis. Such descriptive information may include Plant to Extract ratios, which are ratios of the quantity of botanical article used in the manufacture of the extract to the quantity of extract obtained. Plant to Extract ratios can be misleading when their meaning is not clearly understood.
Botanical extracts are composed of extracted matter obtained from starting materials of botanical origin [United States Pharmacopeia (USP)]. They result from dissolving soluble plant constituents in extraction solvents and separating them from undissolved plant materials. A botanical extract has been defined as &#;the complex, multicomponent mixture obtained after using a solvent to dissolve components of the botanical material&#; (Dentali, ). Crude botanical extracts (those without added excipients) are called native or genuine extracts. Excipients are often added to extracts to improve their material handling characteristics, to standardize constituent concentrations, and for other functional purposes. The term &#;botanical extract&#; here refers to all types of extracts, independent of the relationship between identified constituents and the bioactivity (potency) of the extract.
Although botanical extracts may be subjected to additional processing to enrich the content of a particular chemical class of constituents, once they have been isolated as a fraction or as purified single constituents, these articles and expressed juices are not considered to be botanical extracts. Furthermore, extracts do not include chemically modified plant constituents except where artifacts result from heating or extraction processes. In addition, for the purposes of this article, the term plant or botanical is used in the broad sense to include algae, fungi, and lichens, as well as plant products such as exudates or oleo-gum-resins.
Botanical extracts can be described, in part, by the Plant to Extract ratio of botanical starting crude material (a.k.a. biomass) from which they were made, to the resulting native, or finished, extracts. The European Medicines Agency (EMA) [European Medicines Agency (EMA), ] guidelines refer to the ratio of starting material to genuine/native extract ratio as the DER genuine (Drug to Genuine Extract Ratio),&#; and explain how to disclose the percent of genuine/native extract with the percent excipients for finished extracts. The DER is used as part of the strength characterization of an extract, i.e., the amount of starting material used to make a unit of extract. Community Herbal Monographs from EMA use the DER, together with extraction solvent information, for the determination of crude starting plant material equivalents in comparing extracts, and for estimating extract dosages according to well-established or traditional plant uses.
Plant to Extract ratios may enable the determination of an extract&#;s raw material equivalents, although the final chemical composition can vary depending on the quality of the starting plant material and the extraction conditions. A close comparability of extracts, also known as their phytoequivalence (Australian Government. Department of Health, ), cannot be determined without a detailed comparison of the solvent(s) and manufacturing processes used, sometimes supplemented with comprehensive comparisons of the extracts&#; chemical compositions.
This article first provides an overview of botanical extracts, including their different types and forms, standardization, and categorization. Additional sections explain the concept of Plant to Extract ratios and the common misconceptions. Also provided are recommendations on how to apply the Plant to Extract ratio in labeling, and its relevance to the dosage of plant material equivalents.
Botanical extracts, as defined in the USP General Chapter <565> Botanical Extracts [United States Pharmacopeia (USP)], are often used as ingredients in dietary supplements. Botanical extracts that conform to USP monographs for Botanical Extracts should be obtained from botanical articles that also conform to the corresponding USP monographs. Extracts may be manufactured to concentrate desired constituents, decrease the content of unwanted constituents or impurities, improve shelf life, and produce consistent material for the testing of claimed benefits. Depending on the type of botanical material and extraction technology used, prior to extraction the raw starting material may be subjected to different types of pretreatments, including cutting and grinding to reduce particle size and optimize surface area exposure, defatting, etc.
The composition of botanical extracts from the same plant may vary significantly depending on the extraction solvent(s) used, the temperature and duration of extraction, and the processes used to dry the extracts. Other sources of variation include the steps taken to concentrate or remove targeted constituents or classes of constituents, and the compounds formed during extraction or further processing. Additional variation in the composition of botanical extracts made using the same plant species and plant part as starting materials may occur due to genetic factors, environmental conditions, and agricultural practices. Managing the natural variations in starting material and using standardized extraction procedures can serve to create extracts with consistent composition. Suitable inert substances (excipients) are often added to extracts via granulation or other procedures to act as carriers or diluents which improve physical handling characteristics such as flowability and mixability. Excipients may also facilitate the production of a powder, reduce clumping, and improve homogeneity, bioavailability, stability, and other characteristics. Excipients can also be used to standardize the extract to a defined content of one or more constituents.
According to USP General Chapter <565> Botanical Extracts [United States Pharmacopeia (USP)], botanical extracts can be classified according to their physical state as either liquid (e.g., fluidextracts and tinctures), semisolid (soft), or solid (dry) forms. These physical forms are defined in Table 1.
TABLE 1
TABLE 1. Botanical extract types and forms.
Various organizations define botanical extract standardization differently
The American Herbal Products Association (AHPA) (American Herbal Products Association, ), the leading herbal trade organization in the United States, defines standardization as &#;the complete body of information and controls that serves to optimize the batch-to-batch consistency of a botanical product. Standardization is achieved by reducing the inherent variation of natural product composition through quality assurance practices applied to agricultural and manufacturing processes.&#; AHPA (American Herbal Products Association, ) points out that, &#;[i]n fact, standardization&#;when properly performed&#;entails a lot more than merely controlling the content of a particular marker compound &#; It comprises a wide variety of raw material and process controls, as well as use of a consistent recipe.&#;
Marker compounds are constituents that may or may not be associated with therapeutic activity and often are used as in-process controls. They also can help demonstrate identity when specific to the botanical raw material under consideration. On the other hand, marker compound levels may not vary proportionally with other compounds of greater importance relative to therapeutic activity, due to differences in genetics, growing conditions, or stability during processing and storage.
The EMA categorizes &#;standardised extracts&#; as those where the identified constituents are understood to fully account for an extract&#;s proven therapeutic activity [European Medicines Agency (EMA), ]. The relationship of identified constituents to an extract&#;s biological activity may not always be clear. Awang () noted that the identity of constituents responsible for biological activities of a plant extract are rarely clearly established, even with bioassays and clinical studies, and numerous constituents may be active to different degrees and in various respects. A few examples of bioactive constituents in &#;standardised extracts&#; include the laxative sennosides of senna leaf, the hepatoprotective silymarin flavonolignans of milk thistle fruit, and the anti-nauseant gingerols of ginger rhizome.
Elimination of unwanted constituents, so-called negative markers, from extracts is also considered a form of standardization. Examples of negative markers include the neurotoxic thujones found in tansy, and hepatotoxic pyrrolizidine alkaloids found in comfrey and other herbs. Marker compounds include constituents that are characteristic of a particular species or variety of a plant, and thus are useful for standardization but may not be entirely responsible for the intended therapeutic activity. Examples include parthenolide in feverfew and echinacoside in Echinacea angustifolia and E. pallida.
Bioassays of extracts may provide some measure of therapeutic activity, although they are rarely used for standardization. A classic example is the use of in vivo bioassays with frogs, cats, and pigeons to standardize extracts of digitalis (Dieuaide et al., ; Lehman, ). In another example, bioassay is recommended as a way to ensure reproducible pharmacological activity (potency) of the dragon&#;s blood (Croton lechleri) latex botanical drug (U.S. Department of Health and Human Services, ). Although they may be useful for standardizing extracts to a potency measurement, challenges with bioassay-based standardization include cost, complexity, and demonstrating the relationship to clinically relevant effects in humans.
Standardization to either active or marker constituents and bioassays that reflect the underlying mechanisms of action were described by van Breemen et al. (). Hubbard et al. () reported that recent developments in bioinformatics and bioassay technology have made it possible to address large numbers of phytochemical constituents in a plant extract and the potential diversity of their biological effects. This allows a much greater level of detail for characterizing the features that could be used for plant extract standardization.
In addition to the classification of extracts as to whether they are liquid, soft, or dry, the aforementioned EMA category of &#;standardised extracts&#; is joined by two other categories of extracts, namely &#;quantified extracts,&#; and &#;other extracts,&#; depending on the relationship between known constituents and the extract&#;s biological activity [European Medicines Agency (EMA), ]. Unlike the EMA-designated standardised extracts, in quantified extracts the identified constituents partly, but not fully, account for an extract&#;s bioactivity. In this case, and for &#;other extracts&#; that have no relationship between identified constituents and extract bioactivity, the whole of the extract is considered to be the active material.
Crude extracts may be processed further, or a selective extraction may be performed at the outset to concentrate particular classes of phytochemicals or to decrease the content of unwanted constituents, or both. Gymnema leaf extract containing NLT than 5.0% of gymnemic acids as USP Purified Gymnema Extract, and USP Powdered Ginkgo Extract that contains NLT 22.0% and NMT 27.0% flavonol glycosides, and NLT 5.4% and NMT 12.0% of terpene lactones, are examples of extracts in which specific constituents are enhanced. Licorice root deglycyrrhizinated extract and green tea leaf decaffeinated extract with a concentrated content of catechins as USP Powdered Decaffeinated Green Tea Extract represent extracts in which specific constituents have been removed.
These specialized extracts fall along a chemical complexity continuum from selective and semi-purified botanical extracts or extract fractions that concentrate a phytochemical class, to an isolated class of phytochemical constituents, to a single compound (Figure 1). Depending on the degree of chemical purification, these latter two examples may no longer be appropriately considered extracts. Examples of concentrated constituent classes of compounds include sennosides extracted from senna leaflets or senna pods (USP Sennosides) and curcuminoids extracted from turmeric rhizome (USP Curcuminoids).
FIGURE 1
FIGURE 1. Article of Botanical Origin for Turmeric (Curcuma longa L.) rhizome form intact plant material to single chemical entity [United States Pharmacopeia (USP), ].
It is important to clarify the concept of Plant to Extract ratios because misunderstandings regarding what they signify are common. Plant to Extract ratios reflect the amount of material extracted from plant biomass relative to the starting amount of biomass. They may be used to partly define extracts with or without the presence of added excipients. The calculations of Plant to Extract ratios should be made on the dried basis irrespective of whether the starting raw material used in the extraction is in fresh or dried form.&#;
Similar to the EMA guidelines, the Kooperation Phytopharmaka () defines the plant drug to extract ratio (DER) as the ratio of the amount of starting plant used to produce a certain amount of native extract that is exclusive of any carriers or other excipients. For example, a dry extract with an average native extract ratio of 10:1 means that approximately 10 g of dried raw material were required to produce 1 g of native/genuine extract.
In practice, native extract production yield will usually vary due to the inherent variation of extractive matter from different batches of starting materials; this results in a Plant to Extract ratio range in place of a single ratio. For example, using the same extraction conditions, one 100-kg lot of starting plant material may yield 14 kg of native extract while a different 100-kg lot may yield only 11 kg of extract. The Plant to Extract ratio in this case would be the range of 7 through 9 to 1, expressed as 7&#;9:1 (100 divided by 14 equals approximately 7, and 100 divided by 11 equals approximately 9). Only 7 kg of starting material would be needed to produce 1 kg of native extract in the first instance while 9 kg of another lot of the same plant would be required to produce an equivalent amount of extract.
Extract yields are fundamental to the calculation of Plant to Extract ratios. Perhaps the most common misconception regarding Plant to Extract ratios is that a higher ratio represents a stronger, and therefore better extract. Extract yields depend on the extraction process and the amount of extractable material in the starting plant biomass; Plant to Extract ratios describe the extract yield from a given raw material using a given manufacturing process. Consider a hypothetical case where all the starting material is converted to dry extract. In this case, the extract yield would be 100 percent and the Plant to Extract ratio would be 1:1,§ indicating that each unit of extract represents an equivalent amount of starting material. However, for most dry botanical materials extracted in aqueous or hydroethanolic solvents, the amount of extractable matter (soluble constituents) from the biomass is usually between 10 and 25 percent, which calculates to starting plant mass to dry extract ratios of 10:1 and 4:1, respectively (100 divided by 10 is 10, and 100 divided by 25 is 4).
The Australian Therapeutic Goods Administration (TGA) Guidance on Equivalence of Herbal Extracts in Complementary Medicines (Australian Government. Department of Health, ) states: &#;Whilst a high native extraction ratio is generally reflective of a targeted extraction procedure (i.e., specific components or component classes are selected for), there are instances where a high extraction ratio may simply reflect a partial extraction procedure.&#; For example, if a plant biomass with a potential 25% extractable crude material could have an extract ratio of 4:1 with a given manufacturing process but only yields 10% extractible materials of interest with a different process and leaves behind the other 15%, the extract ratio calculation changes from 4:1 to 10:1.
Low or high Plant to Extract ratios can be explained, in part, by the soluble extractive matter starting value. For example, woody roots may naturally contain relatively small amounts of extractable material and result in relatively high extract ratios even when extracted to exhaustion. According to the Hong Kong Chinese Materia Medica Standards, eleuthero root should contain not less than 3.0% water-soluble extractives and 3.0% ethanol-soluble extractives (using the cold extraction method in both cases) (Chinese Medicine Division and Department of Health, a), thus a theoretical native extract ratio of about 33:1. In contrast, Asian ginseng root should contain not less than 27.0% water-soluble extractives and 22.0% ethanol-soluble extractives (using the cold extraction method in both cases) (Chinese Medicine Division and Department of Health, b), thus a theoretical native extract ratio of about 4:1.
Another example of the challenge of using Plant to Extract ratios to compare botanical extract products on the market is illustrated by the case of Asian ginseng. The USP Asian Ginseng Root and Rhizome monograph sets out quality specifications for the dried roots and rhizomes of Panax ginseng, including minimum concentrations for ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd. However, for dried raw material of a given age, the relative contents of ginsenosides Re, Rf, Rb1, Rc, Rb2, and Rd are significantly higher in the fibrous root portion > rhizome > branch root > main root, while the content of Rg1 is highest in the rhizome > branch root > fibrous root > main root (Pan et al., ). The rhizomes and main roots are often separated from smaller branch roots and fibrous roots in the material of commerce. Thus, a higher Plant to Extract Ratio would be needed using main root and rhizome material to achieve the same levels of the marker ginsenosides compared to extracts of the branch and fibrous roots.
As previously mentioned, high Plant to Extract ratios may also reflect manufacturing processes intentionally designed to capture only a narrow range of native constituents, either through use of a selective solvent for initial extraction or through further processing of crude extracts to concentrate specific constituents. For example, the USP Native Gymnema Extract monograph states that the ratio of starting plant material to extract is about 8:1. In contrast, USP Purified Gymnema Extract, prepared by further processing of USP Native Gymnema Extract, has a ratio of starting material to extract of about 25:1 because about two thirds of the native extract is discarded during preparation. Thus, in the case of the USP Native Gymnema Extract, it is evident that 100 g of starting material yields about 12.5 g of native extract, from which it is possible to create about 4 g of the purified extract (USP Purified Gymnema Extract).
Regarding extracts made from materials that yield high Plant to Extract ratios, the TGA makes this very important point: &#;Consideration should be given to ensuring that these extracts are not marketed in a manner that implies that they are &#;better&#; because they are derived from a larger quantity of raw herbal material. Such marketing would represent a misuse on the part of a supplier and a misunderstanding by customers&#; (Australian Government. Department of Health, ).
Botanical extracts are multi-component mixtures that can be produced to an acceptable consistency but are not usually completely uniform due to raw material variations and differences in manufacturing conditions. A full chemical comparison and/or biological testing may be needed to establish phytoequivalence between extracts so that the extracts can be assumed to be equivalent for all intents and purposes. Plant to Extract ratios that allow the calculation of starting material equivalents may serve as a criterion, along with other factors, to establish equivalence between different extracts (Health Canada, ). This applies only if sufficient manufacturing information about the finished extracts is available. This should include, at a minimum, the native extract concentration, extraction solvents used, and the general extraction procedure including steps applied to concentrate or remove constituents or classes of constituent (Australian Government. Department of Health, ). Ultimately, fingerprint characterization of constituents and quantification of marker or active compounds, as described in different sections of the USP botanical extract monographs, may be needed to fully establish phytoequivalence between extracts.
The Australian TGA Guidance on Equivalence of Herbal Extracts in Complementary Medicines (Australian Government. Department of Health, ) identifies the following as some of the factors that impact the phytoequivalence of extracts: starting material quality, solvent choices, and manufacturing processes including time and temperature. In relation to the solvent system, in cases where the type and amount of solvent used to manufacture a particular extract is the same, TGA states that a limited degree of variation in minor solvent concentration is now considered acceptable. In this way, extracts with small differences in extraction solvent systems may be considered phytoequivalent while excluding other solvent systems that could result in significant variation between extracts (Australian Government. Department of Health, ).
The addition of carriers and other excipients to extracts is another important aspect that should be addressed in the description of botanical extracts. According to the Australian TGA (Australian Government. Department of Health, ), &#;there are also situations where an extract with a high native extract ratio is diluted with a carrier or diluent, for a variety of purposes. The addition of diluents and carriers should always be taken into account when assessing whether two extracts are equivalent.&#;
The importance of differentiating between finished extracts containing excipients and 100% native extracts can be illustrated by considering finished extract ratios. For example, if an average of 4 kg of starting material is required to produce 1 kg of native extract, the average Plant to Extract ratio is 4:1. Adding 1 kg of carrier to each kg of native extract doubles the amount of total finished extract. Whereas the starting material to native extract ratio is still 4:1, the addition of carrier results in each kg of finished extract now containing 0.5 kg of native extract and 0.5 kg of excipient(s). The ratio of Plant to (finished) Extract (that is 50% native) is now 2:1. Without appropriate disclosure of the percentage of native extract or the percentage of excipients, a Plant to Extract ratio of 2:1 for this finished extract could imply a higher extraction yield than the original native extract ratio of 4:1. Therefore, accurate calculations of extract starting material equivalents require access to information regarding the percent of native extract and excipients in the finished extract.
Plant to Extract ratio product labeling is required in some countries and not others, depending in part on the regulatory framework applicable for the finished product, i.e., whether the article is regulated as a food, a supplement, an over-the-counter (OTC) drug product, or a prescription drug product. This section covers ingredient labeling recommendations that are transferable to finished product labeling. Examples of Plant to Extract labeling guidelines from the Uniited States, Canada, and Australia are provided below.
In the United States, if a dietary supplement manufacturer claims that a dietary supplement ingredient meets USP standards, the product is misbranded (and thus unlawful) if it fails to actually meet those standards. The USP Guideline for Assigning Titles to USP Dietary Supplement Monographs (United States Pharmacopeia (USP), ) provides extensive details on the different types of botanical extracts for which monographs have been published, and how USP creates monograph titles that are part of the labeling requirements.
The USP General Chapter <565> Botanical Extracts (United States Pharmacopeia (USP)) in the USP&#;NF and in the USP Dietary Supplements Compendium states the following requirement for extract labeling: &#;Label it to indicate the name of the plant part used; the names of solvents, other than the hydroalcoholic solvents, used in preparation; the content, in percentage, of active principles or marker compounds identified in the individual monograph; and the name and concentration of any added antimicrobial or other preservative. Where active principles are unknown, the ratio of starting material to final product is stated. For semisolid extracts and powdered extracts, the identity and quantity of any added excipient is also indicated. In such cases, the percentage of native extract may also be stated.&#;
USP monographs for botanical extracts include Composition tests for percentage limits of identified active principles or marker compounds; manufacturers may also disclose both the extract ratio and excipient content. The EMA, in the Guideline on Declaration of Herbal Substances and Herbal Preparations in Herbal Medicinal Products/Traditional Herbal Medicinal Products (European Medicines Agency (EMA), ), recommends including the percent quantity of genuine extract, the DER (drug to genuine/native extract ratio) of the extract, and the percent amount of excipients in the declaration of ingredient descriptions, as can be seen in the following example:
Dry extract from Valerian root.
Quantity of the genuine extract: 80% genuine extract.
DER genuine: 3&#;6:1.
Other excipients: 20%
Extraction solvent: Ethanol 70% V/V.
For dry (often referred to as &#;powdered&#;) extracts, Title 21 of the United States Code of Federal Regulations (21 CFR) section 101.36(b) (U.S. Department of Health and Human Services, ) (3) (ii) (C) states that &#;[f]or a dietary ingredient that is an extract from which the solvent has been removed, the weight of the ingredient shall be the weight of the dried extract.&#;
In the case of liquid extracts, 21 CFR 101.36(b) [U.S. Department of Health and Human Services, ] (3) (ii) (B) states that for any dietary ingredient that is a liquid extract from which the solvent has not been removed, the quantity listed must be the volume or weight of the total extract. Information on the condition of the starting material must be stated when it is fresh and may be indicated when dried material was used to make the extract. Information may be included on the concentration of the dietary ingredient and the solvent used. The United States Food and Drug Administration (FDA) provides the following as an example: &#;fresh dandelion root extract, x (y:z) in 70% ethanol, where x is the number of mL or mg of the entire extract, y is the weight of the starting material, and z is the volume (mL) of solvent.&#;
AHPA developed a retail labeling guidance for non-liquid botanical extracts titled Guidance for the Retail Labeling of Dietary Supplements Containing Soft or Powdered Botanical Extracts (American Herbal Products Association, ). This guidance includes carriers and other excipients as part of the quantity of a finished extract, which represents how bulk extracts are bought and sold&#;by total weight. AHPA also provides guidance on the voluntary disclosure of the percent of the native extract when it is listed on the label.
AHPA provides the following convention when manufacturers state extract ratios (American Herbal Products Association, ): &#;the first number shall represent the amount of dried botanical starting material, the second number shall represent the amount of finished total extract (emphasis added). For example, a 4:1 extract is one in which each kilogram (or other unit) of finished total extract represents the extractives from 4 kg (or other unit) of dried botanical starting material.&#; Following this convention, the amount of excipient is included in the calculation of starting material to finished extract.
AHPA offers two options for stating Plant to Extract ratios when lot-to-lot variation is encountered. These options are: 1) stating the range for either the native extract percent or for the extract ratio, or 2) using an average of the range when the range does not vary by more than 20% between the highest and lowest values (American Herbal Products Association, ). Two options also are provided for listing average extract values on a label, namely &#;average x% native&#; or &#;average x:1&#;. In practice, single values given for extract ratios generally represent a shorthand for the actual range. Additional information on extract ratios can be found in AHPA&#;s White Paper: Standardization of Botanical Products (American Herbal Products Association, ) and the Guidance for the Retail Labeling of Dietary Supplements Containing Soft or Powdered Botanical Extracts (American Herbal Products Association, ).
EMA (European Medicines Agency (EMA), ) guidelines provide detailed examples of how to declare Plant to Extract ratios that include disclosure of the percent excipients added to botanical extracts. The disclosure of excipients is directly translated to retail labeling of finished products. Following the earlier example of the Valerian root dry extract, Table 2 describes the correct labeling of a finished product (capsule) containing this ingredient.
TABLE 2
TABLE 2. Labeling of a finished product (capsule) containing Valerian Dry Extract according to EMA (European Medicines Agency (EMA), )
The Natural and Non-prescription Health Products Directorate (NNHPD) in Canada specifies the listing of extract ratios on labels with the quantity of dried material used to make it, with the following as an example: Black Cohosh (6:1 extract) .... 40 mg, (Actaea racemosa) (root) equivalent to 240 mg of Black Cohosh (Health Canada, ). Since the amount of native extract or excipients is not specified, the extract ratio in this case takes into account the total amount of extract, including any added excipients, in order to represent the herb raw material equivalent.
The Medicine Labels Guidance on TG O 91 and TG O 92, version 2.3 from the Australian TGA (Australian Government. Department of Health, ), states: &#;If the active ingredient in your medicine is a herbal preparation, its quantity must be expressed as the: weight of that preparation, and equivalent weight of the herbal material from which it was prepared.&#; Where &#;standardisation&#; is claimed (&#;the process in which the content of a specific chemical constituent(s) has been determined in a herbal material or herbal preparation&#;), &#;then the quantity of the active ingredient must be expressed as: the weight of that preparation, the minimum weight of the herbal material from which it was prepared, and the quantity of standardised constituent(s) in the herbal preparation.&#;
The USP Asian Ginseng Root and Rhizome Dry Extract example demonstrates the effect of the addition of excipients on both the native and final extract ratios. This article is prepared from the dried roots and rhizomes of Panax ginseng C.A. Mey. by extraction with water or hydroalcoholic mixtures. It contains not less than 3.0% of ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd combined, calculated on the anhydrous basis, and may contain other added substances. If ten parts of starting material yields two parts of native extract, a 5:1 ratio of Plant to native Extract is obtained (10 divided by 2). If 0.5 part of excipient is added to the two parts of native extract, then the ratio of Plant to finished Extract becomes 4:1 (10 divided by 2.5 is 4). Best practices for labeling of the finished total extract ingredient according to USP General Chapter <565> Botanical Extracts [United States Pharmacopeia (USP)] and its corresponding communication in the finished product are as described in Table 3.
TABLE 3
TABLE 3. Best practices for labeling of finished product (capsule) according to USP General Chapter <565> Botanical Extracts (United States Pharmacopeia (USP))
Plant to Extract ratios provide an indication of strength relative to starting materials, including those recognized as traditional medicines. Plant to Extract ratios may be used to determine relevant raw material equivalents and form a reasonable basis for strength comparisons of raw materials and extracts. In fact, Health Canada states that: &#;an extract can be partially characterized by its specifications and the ratio of the quantity crude equivalent of the whole herb to the quantity of the extract&#; (Health Canada, ). EMA Community herbal monographs indicate the DER and solvent composition used for the manufacturing process; this DER is used to calculate the dose of the corresponding plant material (daily use) linked to the traditional use (European Medicines Agency, ). When active or marker compounds are known, it is recommended to include the quantity of constituents to confirm the relevant raw material equivalents.
Plant to Extract Ratios also play an important role in marketing authorization. Brand company applicants, whether seeking pre-marketing authorization for licensed Natural Health Products (NHPs) in Canada, listed Complementary Medicine Products (CMPs) in Australia, registered Remedios Herbolarios in Mexico, or registered Traditional Herbal Medicinal Products (THMPs) in the EU or UK, must declare in their quality dossiers the specified quality of each ingredient and the amount of excipients used. The Plant to Extract ratio is used in the efficacy dossier for determining dosage calculation. In most countries, Plant to Extract ratios are required to be disclosed on the label of the finished product.
This article explains the concept and use of Plant to Extract ratios, particularly with respect to dry extracts, and clarifies some of the common misconceptions regarding Plant to Extract ratios and their use. Plant to Extract ratios are important descriptors of botanical extracts linked to the extract yield of a manufacturing process. They may play a role in the estimation of phytoequivalence, labeling of botanical ingredients and corresponding dosage forms, and calculation of the plant material equivalents. To foster accurate communication between suppliers and manufacturers regarding botanical extracts, it is necessary to disclose not only the Plant to Extract ratios, but also to include the complete botanical extract composition, including any excipients and their percentage in the extract, extraction solvents, and the amount of active or marker constituents. In the absence of this information, the use of Plant to Extract ratios to calculate relevant raw materials should be considered with caution.
Plant to Extract ratios are among the descriptors of botanical extracts in the Definition and Labeling sections of USP monographs for botanical extracts. Manufacturers can follow the complete labeling recommendations for botanical extracts as described in <565> Botanical Extracts, which can help manufacturers to create accurate labeling of finished products containing botanical extract ingredients and support consumer decision-making when comparing similar products containing botanical extracts.
JB, SD, SG, RM, MM, and HO-R: Prepared the manuscript draft. TB, GG, HJ, JK, CM, PP, and NS: Provided documents, practical examples, technical comments and discussions. All: Contributed to review and editing.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
*As per Title 21 of the Code of Federal Regulations (CFR) Sec. 111.70(b). Following 21 CFR Sec. 111.105, a dietary supplement company&#;s quality control personnel are responsible for approving or rejecting specifications.
&#;In this paper, the meaning of the term &#;Plant to Extract Ratio&#; is equivalent to &#;Drug to Extract Ratio (DER)&#; (used in Europe) and &#;Extract Ratio&#; (used in Canada and Australia). The term &#;herbal drug&#; as defined in the European Pharmacopoeia (also referred as &#;herbal substance&#; by the European Medicines Agency) is known as a botanical dietary ingredient as defined by the United States Food and Drug Administration (FDA), and hence the use of the term Plant to Extract ratio under the United States regulatory framework for Dietary Supplements.
&#;A dried botanical material is that which is in conformance with the Loss on Drying test in individual USP monographs.
§In the case of liquid extracts, a 1:1 ratio defines fluidextracts.
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For more information, please visit China Mango Powder Wholesale in Bulk.
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Herbs have been used for centuries in order to enrich food as preservatives, flavorings, and medicinal agents. The aim of this work was to study the effect of the addition of selected herbal extracts (dried leaves of Thymus vulgaris L., Origanum vulgare L., Satureja hortensis L., Rosmarinus officinalis L., and Ocimum basilicum L.) on selected parameters of fermented flavored cream (counts of starter culture bacteria and pH value) and the resulting flavored butter (water content, water distribution, butter plasma pH, butter fat acidity, and oxidative stability), preceded by a study of the activity of the herbal extracts against starter lactic acid bacteria determined using the well diffusion method. The extracts did not inhibit the starter lactic acid bacteria at a fixed level. The presence of the herbal extracts contributed to a shorter fermentation course and influenced the counts of starter culture bacteria during fermentation and refrigerated storage (at 5 °C) for 21 days. The extract additives did not affect the water content or the degree of its dispersion, the butter plasma pH, or the butter fat acidity. The positive effect of the rosemary and thyme extract addition was only noted when analyzing the oxidative stability of the milk fat of the butter.
The aim of this work was to study the effect of the addition of selected herbal extracts on selected quality characteristics of fermented flavored cream and the resulting flavored butter. We aimed to study the effect of the addition of the whole extract of the selected herbs, but not the specific compounds isolated from them.
Milk and dairy products are unique carriers successfully used to deliver the bioactive ingredients that benefit human health. Furthermore, the addition of herbs and spices, or their extracts, to various dairy products enables these products to function as carriers of nutraceuticals [ 1 , 2 ].
In recent decades, consumer requirements for food production have changed significantly. Today, food is designed not only to satisfy hunger and provide people with essential nutrients, but also to prevent diet-related diseases and improve people&#;s physical and mental well-being. Meanwhile, herbs have been used for centuries to enrich food as preservatives, flavorings, and medicinal agents [ 1 , 2 ]. Today, herbs and spices are used as food additives around the world, not only to improve the organoleptic properties of food, but also to extend shelf life. Due to their antimicrobial and antioxidant properties, they are useful in the dairy industry as a flavor fixative [ 3 ]. It has become apparent that lipids can be effectively protected from enzymatic hydrolysis, oxidation, and other adverse transformations by a variety of natural spices, sometimes more effectively than by synthetic antioxidants. Therefore, the addition of herbal spices to butter may now become a popular way to introduce safe antioxidant substances into this fat. Previous studies on the isolation of oregano (Origanum vulgare L.) essential oils from various regions of the world have focused on the chemical composition, although the antioxidant and antimicrobial properties have also been studied [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. Herbal extracts have powerful antioxidant and antimicrobial effects, mainly due to the amount and quality of the phenolic compounds they contain [ 5 , 7 , 8 , 10 ]. Savory (Satureja hortensis L.) herb has significant antimicrobial activity, which is linked to the presence in the herb of an oil rich in carvacrol, a phenolic component that exhibits strong antimicrobial activity [ 13 , 14 ]. Thyme herb (Thymi herba) exhibits a broad spectrum of activity against the growth of many strains of Gram-positive and Gram-negative bacteria [ 15 , 16 ]. Common basil (Ocimum basilicum L.) extract could quench DPPH+ radicals and result in a high degree of inhibition of linoleic acid oxidation [ 17 , 18 ]. In addition, rosemary (Rosmarinus officinalis L.) is a plant rich in compounds that exhibit biological activity [ 3 , 19 ].
Calculations of the mean and standard deviation values of the results were performed using an MS Office Excel spreadsheet. One-way or two-factor ANOVA analysis of variance was used for the statistical analysis of the obtained results in Statistica (version 13.3, StatSoft, Krakow, Poland). Comparisons of the significance of differences between the obtained results were made based on Tukey&#;s test (HSD) at the significance level α = 0.05.
Oxidative stability. The butter samples were subjected to Rancimat accelerated oxidation test conditions, according to Gramza-Michalowska et al. [ 5 ], using a Rancimat-type 743 apparatus (Metrohm AG, Herisau, Switzerland). In the reaction vessel, a 3-g sample of butter fat phase was oxidized at 120 °C (air flow 20 dm 3 /h). The end of the induction period was characterized by a quick increase in water conductivity (due to the dissociation of volatile carboxylic acids). The analysis was conducted in duplicate.
Determination of butter fat acidity. Ten grams of butter fat were weighed into Erlenmeyer flasks, then 25 mL of neutralized ethanol and 2 drops of 2% alcoholic phenolphthalein solution were added. Next, the content was heated to boiling and shaken vigorously, followed by titrations with 0.1 N NaOH until a slightly pink color was obtained that lasted for 30 sec. The analysis was conducted in duplicate. Fat acidity was expressed in degrees (°K) as a volume (mL) of sodium hydroxide solution of molar concentration (0.1 mol/dm 3 ), spent for the neutralization of free acids in 10 g of product [ 24 ].
The pH value of the butter plasma. The test consisted of gently melting 40.0 g of butter sample in a water bath at 40 °C, then separating the aqueous phase from the fat phase using an MPW-350R centrifuge (MPW Med. Instruments, Warsaw, Poland) at × g for 15 min at 40 °C. The fat separated on top was carefully collected for further analysis. The aqueous phase of the butter (butter plasma) was analyzed to measure the pH. The pH value of the butter plasma samples was measured at 25 °C using a CPO-505 pH meter (Elmetron) with a conventional electrode. The analysis was conducted in duplicate.
The main principle of the method of water distribution in the butter is to apply indicator paper saturated with indicator to the freshly cut butter surface [ 23 ]. The analysis was performed using commercial indicator paper (Dysperwod, LABLACTA, Olsztyn, Poland) according to the manufacturer&#;s instructions. This method allows for the determination of whether the butter has been properly kneaded and the water droplets properly dispersed in the butter matrix. The indicator paper turns dark blue where it encounters water droplets. To determine the degree of water distribution, a point scale of 0&#;3 was used, according to which the products were classified using the criteria given in . The analysis was conducted in duplicate.
Determining the water content in the butter consisted of establishing, by weight, the weight loss of the sample dried with properly prepared sand and calculating the percentage of water content in the product [ 22 ]. A weight of approximately 4 g of butter, mixed with sand, was dried in a laboratory dryer at 102 °C for 3 h, then cooled and redried for another hour at the same temperature. The drying process was conducted until there was no loss of weight of the dried sample. The analysis was conducted in duplicate.
The presence of contaminating microflora was measured after butter manufacture and during its refrigerated storage. The total number of Enterobacteriaceae was determined in VRBG medium (Merck) overlaid on the same medium and incubated at 37 °C for 24 h. The total numbers of molds and yeasts were determined in YGC medium (Merck), and the plates were incubated at 25 °C for 5 days. The result was given in colony-forming units of 1 mL (cfu/mL). The analysis was conducted in duplicate.
The resulting butter samples were also stored at 25 °C and 5 °C for 21 days for further analysis. The water content and the degree of water dispersion were examined in the resulting butter. The butter samples were stored in portions of 70 g in sterile glass jars (tightly sealed and away from light). Microbiological (contaminating microflora) and physicochemical (water content, water distribution, pH value of the plasma, fat acidity, and oxidative stability) analysis of the butter samples was conducted at 0, 7, 14, and 21 days of sample storage.
The freshly fermented cream was subjected to the buttering process in a laboratory butter churner machine type 83 (Zelmer, Warsaw, Poland). The fermented cream was brought to a temperature that would allow the buttering process to proceed (at 8 °C), and the buttering process was conducted in a butter churner machine until butter grains formed (approximately 45&#;60 min). The buttermilk was then removed from the churner machine, and the butter grains were rinsed twice with cold, previously pasteurized drinking water (the time of each rinse was approximately 3 min at 8 °C). After the rinse water was removed, the butter was subjected to the kneading process, which removed the excess free rinse water, leading to the homogeneous dispersion of water in the fat phase. The experiment was conducted in duplicate.
At the same time as measuring the counts of starter culture bacteria, the presence of contaminating microflora was measured. The total number of Enterobacteriaceae was determined in VRBG medium (with crystal violet, neutral red, bile salts, and glucose, Merck) overlaid on the same medium and incubated at 37 °C for 24 h. The total numbers of molds and yeasts were determined in YGC medium (yeast extract glucose chloramphenicol agar FIL-IDF, Merck), and the plates were incubated at 25 °C for 5 days. The result was given in colony-forming units of 1 mL (cfu/mL). The analysis was conducted in duplicate.
Every two hours of the fermentation of the cream and every seven days of the refrigerated storage of the fermented cream, samples were taken to determine the counts of starter culture bacteria. The bacterial counts of the starter cultures were determined in the: M17 agar (BioMaxima, for Streptococcus thermophilus); De Man, Rogosa and Sharpe agar (MRS agar, BioMaxima, for Lactobacillus spp.); MRS-CC agar (BioMaxima, with clindamycin at 0.5 mL/L and ciprofloxacin at 5.0 mL/L, for Lactobacillus acidophilus); and BSM agar (Fluka, with BSM Supplement, for Bifidobacterium lactis) using the droplet method [ 21 ]. The Petri dishes were incubated at 37 °C for 72 h under appropriate aerobic conditions, depending on the microorganism (Petri dishes with MRS agar, MRS-CC agar, and BSM agar under anaerobic conditions in anaerobic culture containers; Petri dishes with M17 agar under aerobic conditions). After the incubation was completed, the grown colonies were counted. The result was given in colony-forming units of 1 mL (cfu/mL) and then expressed as the logarithm of the total number of bacteria cells. The analysis was conducted in duplicate.
During the fermentation of the cream, samples were taken every hour to measure the pH value. The pH value of the cream samples was measured at 25 °C using a CPO-505 pH meter (Elmetron, Zabrze, Poland) with a conventional electrode probe [ 21 ]. The instrument was calibrated using buffer solutions of pH 4.00 and 7.00 at 25 °C. The analysis was conducted in duplicate.
The cream was fermented in portions of 150 g in sterile glass jars. Before fermentation, 1.5 mg tested lyophilized herbal extracts and 1 mL sterile suspension of starter cultures (previously hydrated for 20 min by dissolving 5 g lyophilizate in 10 mL sterile Ringer&#;s solution) were added to each cream portion. A control sample was also prepared for each variant of the starter culture, which did not contain the addition of herbal extract. The cream samples prepared in this way were kept in an incubator at 45 °C for 6 h to ferment the cream. After the fermented cream manufacture, the samples were left in the refrigerator at 5 °C for 21 days for further analysis. Microbiological and physicochemical analysis of the fermented cream samples was conducted at 0, 7, 14, and 21 days of sample storage. The experiment was conducted in duplicate.
The agar well diffusion method is widely used to evaluate the antimicrobial activity of plant extracts and microbial extracts [ 4 , 6 , 8 , 9 , 10 , 11 , 12 ]. The surface of the medium plate was inoculated by spreading 1 mL of inoculum of starter culture bacteria (hydrated from lyophilized starter culture and revived after overnight incubation in nutrient broth, whereby the concentration of starter culture microorganisms was approximately 7&#;8 log cfu/mL) over the entire surface of the medium. Next, holes with a diameter of 6 mm were cut aseptically with a sterile cork borer, and 20 µL of herbal extract solution at the desired concentration was introduced into the well. The herbal extract solution was prepared in two versions: in sterile DMSO (dimethyl sulfoxide) and in a sterile 68% aqueous ethanol solution. Solutions were prepared by weighing 0.01 mg of each lyophilized herbal extract into 1 mL of each solvent. In order to perform the experiment, previously prepared Petri dishes with appropriate medium were prepared: M17 agar (BioMaxima, for Streptococcus thermophilus); De Man, Rogosa, and Sharpe agar (MRS agar, BioMaxima, for Lactobacillus spp.); MRS-CC agar (BioMaxima, with clindamycin at 0.5 mL/L and ciprofloxacin at 5.0 mL/L, for Lactobacillus acidophilus); and Bifidus Selective Agar (BSM agar, Fluka, with BSM supplement, for Bifidobacterium lactis) [ 21 ]. The agar Petri dishes were then incubated at 37 °C for 72 h under appropriate aerobic conditions, depending on the microorganism (Petri dishes with MRS agar, MRS-CC agar, and BSM agar under anaerobic conditions in anaerobic culture containers; Petri dishes with M17 agar under aerobic conditions). The antimicrobial substances present in the tested extracts diffused in the agar media and inhibited the growth of the tested microorganisms, and after incubation in the Petri dishes, the zones of inhibition (mm) of bacterial growth around the excised wells were measured with a digital caliper. The analysis was conducted in duplicate.
The raw material in the experiments was fresh Łowicka UHT cream with a fat content of 36% (OSM Łowicz, Łowicz, Poland). It was fermented using two industrial lyophilized dairy starter cultures: YC-X16 (from Chr. Hansen, containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) and YO-MIX 207 (from Danisco, containing Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, and Bifidobacterium lactis). All chemicals used were of chemical purity and were purchased from BioMaxima (Lublin, Poland), Fluka (Merck KGaA, Darmstadt, Germany), or Merck (Merck KGaA, Darmstadt, Germany).
The material for the study included extracts of commercially available dried leaves of popular herbs: thyme (Thymus vulgaris L.), oregano (Origanum vulgare L.), savory (Satureja hortensis L.), rosemary (Rosmarinus officinalis L.), and basil (Ocimum basilicum L.). The extracts were prepared by extracting dried leaves according to the protocol previously described in Kozłowska et al. [ 20 ], with some modifications. The preparation of the extract from the different herbs was the same. In general, per 10 g portion of dried herb leaves, an amount of 250 mL of 70% aqueous ethanol solution was used (it is noteworthy that ethanol is an environmentally friendly and safe solvent for human and is well suited for the extraction of phenolic compounds). This mixture was heated for 10 h in a water bath at 45 °C. Next, the solid plant residues were drained on Whatman No. 1 filter paper, while the ethanol was evaporated under vacuum in a Rotavapor R-200 rotary evaporator (Büchi Labortechnik, Flavil, Switzerland). The procedure for herbal extracts manufacture was carried out until sufficient quantities of lyophilized extracts had been collected to carry out the experiments described below. The resulting herbal extracts were lyophilized (Alpha 1-4 LSCplus, Osterode am Harz, Germany) and then stored tightly closed at &#;21 °C in a darkroom until used in experiments.
Herbs and spices are natural ingredients, widely used as a food additive. The use of herbal extracts in dairy production can increase the variety of dairy-based products offered and may supply additional benefits. We aimed to study the effect of the addition of the whole extract of the herbs, but not the specific compounds isolated from them. We realized that the effect on the basis of specific compounds could be completely different from the use of the whole herbal extract. However, the activity of the antioxidant and antimicrobial components present in herbs makes it necessary to verify various aspects of their effects, with many technological challenges faced when developing dairy products enriched with herbs and spices. In this manuscript, we have focused on the effects of selected herbal extracts (as a whole), obtained with ethanol, and we hope that this will contribute to further research on individual compounds of these extracts showing antioxidant, antimicrobial or other interesting effect on the quality or stability of the dairy product.
From a dairy technology perspective, it is important not only to inhibit contaminating or pathogenic microflora, but also to ensure that the extracts do not affect the technical microflora.
The size of the zones of inhibition of bacterial growth with both the 68% ethanol and the DMSO aqueous solutions did not exceed 1 mm ( ). This means that the tested extracts of savory, basil, oregano, rosemary, and thyme did not inhibit the lactic fermentation bacteria included in the tested cultures at the set concentration. Most importantly, our earlier scientific studies confirm the results obtained in the present study [8,25]. Kozlowska et al. [25] conducted research on the effect of coriander essential oil on the growth of lactic acid bacterial strains. After analyzing the values of the average zones of growth inhibition of the tested strains of lactobacilli, it was found that they varied depending on the type and concentration of oil used and the bacterial strain used. As the concentration of added coriander essential oils increased, a larger zone of growth inhibition of the tested Lactobacillus bacteria was observed. This may favor the use of these oils for Lactobacillus products. Furthermore, a study by Piasecka-Jóźwiak et al. [26] showed that thyme oil had the strongest negative effect on all the bacterial strains they evaluated. A slight increase in the concentration of thyme oil to 0.1% in the MRS agar resulted in a decrease in the number of bacterial cells below the baseline level. Of all the LAB (lactic acid bacteria) assessed, only Lactobacillus lactis subsp. lactis KKP 835 was characterized by its ability to grow under these conditions. The highest concentration of thyme oil that did not affect bacterial growth for this strain was 0.1% (v/v).
It transpires that, in terms of the effect of extracts on starter culture microflora, plant extracts show high selectivity. For example, an extract from the leaves of densiflora pine (Pinus densiflora) showed activity against Clostridium perfringens, Staphylococcus aureus, and Escherichia coli, without inhibiting the growth of beneficial microflora such as Lactobacillus and Bifidobacterium [3,11,25]. Diniz do Nascimento et al. [27] showed the inhibitory effect of the essential oils of clove, cinnamon, and rosemary on some species of Lactobacillus, but Ali et al. [28] already observed no inhibitory effect of rosemary on the growth of L. plantarum. However, Saguibo and Elegado [29] noticed the resistance of the probiotic lactobacilli against some plant extracts, including avocado (Persea americana Mill.) and malunggay (Moringa oleifera Lam.) leaves.
The resulting pH measurements taken during the 6-h fermentation of the cream are shown in . The results of the pH measurement taken during the 6-h fermentation of the cream with the YC-X16 culture are shown in a and the initial pH of the cream averaged 6.69 ± 0.13. Meanwhile, the results of the pH measurement performed during the 6-h fermentation of the cream with the YO-MIX 207 culture are shown in b and the initial pH of the cream averaged 6.69 ± 0.12. The samples initially did not deviate from the original pH, regardless of the additive and the type of herbal extract used.
Open in a separate windowFrom the data presented in , it can be seen that the control sample, which did not hold any of the tested extracts, statistically significantly differed from the other samples with the addition of the savory, rosemary, oregano, basil, and thyme extracts, which are determined as constituting one homogeneous group. Statistical analysis distinguished two homogeneous groups, one of which included the cream samples containing the oregano, basil, thyme, rosemary, and savory extracts, and the other the control sample of cream. This means that the addition of the tested extracts at a fixed level did not inhibit the fermentation of the cream, and their presence contributed to a shorter fermentation course. In turn, grouping the results of the two-factor analysis of variance according to fermentation time resulted in the determination of six homogeneous groups. While later measurements were taken, significant differences in pH changes were noted only after the 5th hour as fermentation slowed down, and the results were not significantly different from each other.
Comparable results were obtained by Amirdivani and Baba [30], who showed that herbal extracts improved milk fermentation through yogurt bacteria and increased the acidity of yogurts. Khelif et al. [31] conducted a study to investigate the effect of thyme extract on LAB growth and concluded that a 2% and 4% addition of thyme extract can be added to yogurts without the risk of deterioration in physicochemical quality or inhibition of lactic bacteria. Bakrm and Salihin [32] found that adding aqueous extract of Ceylon cinnamon and common garlic to goat&#;s milk, cow&#;s milk, and camel&#;s milk had no negative effect on acidification through fermentation. Other studies report that the addition of moringa, peppermint, fennel, or common basil extract to yogurt reduces fermentation time by increasing the growth of yogurt cultures [33].
The results presented in show the survival of the microflora during the fermentation process of cream via the YC-X16 starter culture ( a) and the YO-MIX 207 starter culture ( b), and with addition of tested extracts. From the presented data, it can be seen that the addition of almost all of the tested herbal extracts to the cream had a significant (p < 0.05) effect on the cell counts of bacteria of the S. thermophilus species, as well as a selective influence on the genus Lactobacillus, bifidobacteria, and the L. acidophilus species. The statistical analysis confirmed these observations (p < 0.05). When comparing these results with the pH measurements, we found no clear correlations. For bacterial cell counts, the addition of herbal extracts sometimes stimulated a higher number of cells than in the control sample (e.g., L. acidophilus in the cream with the YO-MIX 207 culture), and in some samples, the control sample contained a higher bacterial counts than the other cream samples (e.g., lactobacilli or S. thermophilus in the cream with the YC-X16 culture, and bifidobacteria in the cream with the YO-MIX 207 culture). In addition, by comparing the changes in lactobacilli counts in the cream samples with the YC-X16 culture and the YO-MIX 207 culture, it can be concluded that the effect of the addition of herbal extracts on lactic acid bacteria is dependent on the choice of bacterial strains. These changes were not reflected in the pH changes described above.
Open in a separate windowResearch reports on the effect of plant extracts on the survival of yogurt cultures during fermentation show major differences in results. They vary depending on the bacteria, the composition of the starter culture, the type and amount of extract added, its concentration, and its sensitivity to low pH, among other factors. Some of the data from the literature overlap with the results of the present study, but there are also many discrepancies that require further analysis [34,35,36,37,38,39]. First, the bacterial cell counts in yogurt often decreases below the recommended level (more than 6.0 log cfu/g) [40] due to low pH, high oxygen concentration, increased redox potential, and increased hydrogen peroxide concentration [41,42,43,44,45], making it even more difficult to correctly interpret the effect of the herbal extract addition on the counts of lactic acid bacteria. During fermentation, intensive transformations of milk components take place. Furthermore, the higher buffering capacity of fermented milk beverages can counteract the negative effects of an acidic environment on the counts of lactic acid bacteria and bifidobacteria [38]. The results coinciding with ours were obtained by Arslan et al. [34], who found that yogurt samples with basil and coriander essential oils had lower lactic bacteria counts than the control samples, and, in contrast, the samples with savory oil showed higher lactic bacteria counts than the control samples. Marhamatizadeh et al. [37], on the other hand, showed a positive relationship between growth in L. acidophilus and B. bifidum and the level of olive leaf extract addition. Moreover, Joung et al. [39], studying the properties of herbal yogurt with traditional Korean lotus nut plant extracts, observed an increase in the viability of starter culture bacteria. In contrast, Behrad et al. [36] reported that yogurt mixed with cinnamon and licorice herbs had lower numbers of L. bulgaricus and S. thermophilus live cells compared with the control yogurt.
The results obtained in this study indicated that molds, yeast, and Enterobacteriaceae were found to be absent in all samples during the fermentation of the cream.
The aim of the study is to investigate the effect of adding selected herbal extracts on the viability of the starter culture bacteria, since they were present as viable cells in the final product, which determined the dietary value of the fermented flavored cream. It was interesting to see if the addition of selected herbal extracts affected the counts of viable cells of starter culture bacteria during fermented flavored cream storage. According to the data presented in , the addition of each of the tested herbal extracts to the cream had a different effect on the bacterial cell viability during the 21 days of storage at 5 °C. Statistical analysis also showed that the refrigerated storage conditions of the cream with the herbal extract additives caused significant (p < 0.05) changes in the viability of lactic acid bacteria cells. In general, the greatest changes and the most statistically significant (p < 0.05) impact of the herbal extract additives were observed for the lactobacilli counts in the cream samples fermented with the YC-X16 culture during the 21 days of storage at 5 °C ( a). The largest statistically significant changes in lactobacilli counts during the 21 days of storage at 5 °C were observed in the savory extract cream samples fermented with the YC-X16 culture. An analysis of variance showed that there were significant changes in the number of cells of the microorganisms in question during successive days of refrigerated storage, relative to the number of cells obtained immediately after the fermentation of the cream.
Open in a separate windowOpen in a separate windowThe stimulating effect of plant extract additives was observed by Marhamatizadeh et al. [37], who studied the effect of olive leaf extract on the growth and viability of L. acidophilus and B. bifidum in probiotic milk and yogurt after 21 days of refrigerated storage. The researchers used a 2%, 4%, and 6% addition of olive leaf extract. The results showed that the counts of L. acidophilus and B. bifidum were higher than in the control sample when olive leaf extract was used, and there was a positive relationship between bacterial growth and the level of extract addition. In contrast, according to Arslan et al. [34], the counts of live lactic acid bacteria cells in yogurt samples with basil and coriander essential oils was lower than in the control samples, while in yogurt samples with savory oil, it was higher. Our research confirms the observations made by Joung et al. [39], who showed that the herbal extract and storage time affected several properties of the yogurt, changing the viability of the starter culture. Studies by other authors [35,36,38] show that the effect of adding extracts on yogurt culture viability depends on the type of plant from which the extract is obtained, as well as on the concentrations of phytochemicals present in the extracts used.
Molds, yeast, and Enterobacteriaceae bacteria were all found to be absent in the fermented cream samples until the conclusion of the storage period at 5 °C. This indicates that the observed effects of the herbal extracts used were only related to the starter microflora.
Typically, butter should contain up to live cells of non-pathogenic microorganisms in 1 g [46], although this may be a higher value for butter derived from fermented cream [47]. The use of a step in which butter lumps are rinsed with microbiologically pure water means that butter carries a low microbial load [46]. In addition, proper hygienic production conditions, the right level of water content, and a high degree of water dispersion mean that butter is not a favorable environment for the growth of microorganisms [46,48].
The effect of the addition of the tested plant extracts to the fermented cream was also studied in the resulting butter (the butter was obtained from freshly fermented cream without refrigerated long-term storage). The measured water content was in the range of 15.9&#;16.0% ± 0.1, regardless of the addition of the herbal extract or the starter culture used to ferment the cream. According to the Codex Alimentarius [49], the water content in butter must not be greater than 16%. The results of the present study indicated that the obtained butter samples met these conditions.
The tested butter samples received the maximum number of points (class 3 according to ) when determining the degree of water dispersion. No spots present on the indicator paper were noted on the fresh surface of any of the butter samples, regardless of the addition of the herbal extract or the starter culture used to ferment the cream.
The degree of water dispersion is important from a microbiological perspective [50]. The water phase present in the butter samples allowed optimum conditions for the proliferation of this microflora and the deterioration in the organoleptic qualities of the product, especially at the long-term storage stage. Therefore, the greater the degree of water dispersion, the more difficult the development of undesirable microflora.
An important stage in the production of butter, which subsequently affects the pH of the aqueous phase, is the biological maturation of the cream, i.e., its fermentation [51]. Moreover, the transformations in the aqueous phase of butter are most easily seen by observing changes in the pH of the plasma. depicts that the plasma acidity of the analyzed butter samples obtained similar values to each other, regardless of the addition of the herbal extract or the starter culture used to ferment the cream. This means that the herbal extract additives used had no statistical effect (p < 0.05) on the plasma pH of the butter samples obtained. In addition, such changes were not observed in the butter samples stored at different temperatures: refrigerated (5 °C) and room (25 °C).
From the data presented in , it can be seen that the addition of herbal extracts had no statistically significant effect (p < 0.05) on the acidity of the milk fat. The values found for this acidity were indistinguishable, regardless of the type of herbal extract added, the cream fermentation culture used, or the storage temperature of the butter samples. The only samples whose acidity changed unfavorably during storage were the control samples of butter (without added herbal extracts) kept at 25 °C for 21 days.
In determining the storage stability of butter, the acidity of butter fat is an expression of the amount of free fatty acid and the degree of lipolysis [24,52,53]. Our results were similar to those reported by Trawińska [54], who evaluated that the acidity of butter fat is a result of how the product is stored. A study by Trawińska [54] examined how storage at &#;8 °C, 0 °C, and 20 °C affected the acidity of butter fat. Negative temperatures had no effect on the acidity level of butter. While at 20 °C, it was shown that the acidity up to day 14 was at a similar level, and then increased. This proves that storage temperature is a key factor affecting the overall quality characteristics of butter. The cited researcher showed that negative quality characteristics can be observed when butter is kept at 20 °C for long periods. In our study, statistical analysis demonstrated that the use of herbal extracts had a significant effect on the fat acidity of butter compared with the control samples.
Molds, yeast, and Enterobacteriaceae were all found to be absent in the obtained butter samples with the herbal extract additives until the conclusion of the storage period at 5 °C. This proves the high standard of hygiene in the production of the butter, as well as the positive impact of the used starter cultures on the microbiological quality of the resulting products [55].
The effect of the addition of the herbal extracts was only noticed when analyzing lipid stability under Rancimat test conditions. This analysis allowed an evaluation of the induction period based on the increase in water conductivity caused by the oxidation process [56,57]. shows the duration of the oxidative stability measurement of the tested samples of butter fat obtained from cream with the addition of the tested herbal extracts.
Even immediately after butter manufacture, its samples were characterized by varying induction periods. Statistically significant effect (p < 0.05) on the extension of its value had the rosemary extract and thyme extract additions, in the other cases of additives. The values of the induction period did not differ from the value obtained for the control sample, regardless of the type of starter culture used for the fermentation of the cream intended for butter production.
The oxidative stability of milk fat was also measured after 21 days of storage at 5 °C and 25 °C to compare the effect of added plant extracts on the shelf life of butter over time. After storage at 25 °C for 21 days, as in the case of the measurement immediately after butter manufacture, the longest induction time was characterized by the milk fat samples with the rosemary and thyme extracts. Based on an analysis of variance, it was found that the other induction time results were not statistically significantly different from each other (p < 0.05). Analogous results were obtained for the samples stored at 5 °C for 21 days, which proved the positive effect of the addition of rosemary and thyme on the period of milk fat induction.
The storage conditions also had a significant impact on butter stability [53,58]. As expected, the oxidative stability of the milk fat obtained was higher for the samples of butter kept at 6 °C compared with those kept at 25 °C.
Spices and herbs are known to have enormous potential as natural antioxidants in food, and the general principle of antioxidants is their reaction with oxidizing agents&#;free radicals. Kozowska et al. [59] showed that herbs from the Lamiaceae family (including thyme, oregano, rosemary, lemon balm, savory, hyssop, sage, narrow-leaved lavender and clary sage) have potent antioxidant activity, mainly due to the phenolic compounds present in them. These include eugenol, carvacrol and thymol. This is supported by the study by Bandoniene et al. [60]. Rosemary extract (Rosmarinus officinalis) is one of the most used extracts for this purpose. Its strong antioxidant properties are attributed to its high phenolic component content [8,59]. These properties may even be many times stronger than those found for synthetic antioxidants such as BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) [61,62,63,64]. Médici Veronezi et al. [65] showed an increase in the oxidative stability of thermo-oxidized soybean oil using the example of basil ethanol extract. It is worth noting that the same compounds are responsible for the bactericidal or bacteriostatic properties of herbal extracts. In addition, Kozlowska et al. [59] found that aqueous ethanolic extracts of thyme, rosemary and sage contained slightly higher levels of phenolic compounds compared to aqueous methanolic extracts.
Some authors have also reported the antimicrobial and antioxidant effects of extracts and essential oils of herbs on butter [66,67]. The results of Ayar et al. [68] indicate that methanolic extracts of sage, rosemary, and oregano have a significant effect on butter stability, especially at the 0.05% level. The most effective antioxidants used were sage extract, a sage-rosemary combination, and rosemary extract. Oregano extract and its combinations with rosemary and sage extracts have shown pro-oxidant activity. In addition, Gramza-Michalowska et al. [5] observed that rosemary extract was also an active antioxidant, allowing lipid stabilization two times longer compared with butter control samples. The results of our research are consistent with the results of Gramza-Michalowska et al. [5]. Thus, natural spices and their combinations can be used to increase the oxidative stability of butter.
To conclude, herbs and spices are natural ingredients widely used as food additives. The addition of herbs and spices to dairy products for health benefits should meet requirements in terms of safety, performance, price, and release to avoid any side effects. The use of carefully selected extracts will create products with natural health-promoting properties, with the effect of stabilizing quality parameters.
The main reason for the shelf-life limit of butter is changes in the lipid fraction. They progress as butter is stored and are the result of oxidation and the lipolytic activity of microbial and milk-derived enzymes [5,52]. Regarding the experimental results obtained, it can be concluded that the tested solutions of the savory, basil, oregano, rosemary, and thyme extracts do not inhibit the growth of the lactic fermentation bacteria included in the industrial starter cultures.
To summarize, the present study indicates the strong antioxidant activity of the selected examined herbal extracts in lipid systems. However, Kozłowska et al. [8] found no relationship between the phenolic content of spice extracts and their ability to inhibit LAB growth. Of the plant extracts evaluated, the rosemary and thyme extracts had a significant beneficial effect on the oxidative stability of butter. The other plant extracts had no statistically significant effect on the oxidative stability of butter. No significant effect of storage temperature was observed on the obtained results. Therefore, plant extracts can be successfully used as a functional additive to protect the lipid fraction of fermented foods.
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