Development of A Functional Beverage

International Journal of Food
Engineering
Volume 7, Issue 3 2011 Article 1
Development of a Functional Beverage

Formulation with High Protein Content, Inulin
and Stevia
Laura T. Rodriguez Furlán, INTEQUI-CONICET-UNSL

Antonio Pérez Padilla, Universidad Nacional de San Luis
Mercedes Campderros, Universidad Nacional de San Luis -
CONICET
Recommended Citation:
Rodriguez Furlán, Laura T.; Pérez Padilla, Antonio; and Campderros, Mercedes (2011)
"Development of a Functional Beverage Formulation with High Protein Content, Inulin and
Stevia," International Journal of Food Engineering: Vol. 7: Iss. 3, Article 1.
DOI: 10.2202/1556-3758.2014
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Development of a Functional Beverage
Formulation with High Protein Content, Inulin
and Stevia
Laura T. Rodriguez Furlán, Antonio Pérez Padilla, and Mercedes Campderros
Abstract
A beverage formulation with proteins of high quality, inulin, Stevia and C vitamin has been
prepared. The protein source was bovine plasma which was concentrated and demineralized by a
combined membrane methodologies as microfiltration, ultrafiltration and discontinues
diafiltration. The use of inulin as protective agent to prevent protein concentrate denaturation
during freeze-drying was assayed. The inulin is a versatile food ingredient and it also has health
benefits. The powder obtained has improved sensory characteristic and high solubility. Stevia has
been added as sweetness, being a low-carbohydrate, low-sugar food alternative. The formulation
could be destined as protein supplement with functional properties or for special dietary use.
KEYWORDS: plasma protein, ultrafiltration, functional beverage, inulin, Stevia.

Rodriguez Furlán et al.: Development of a Functional Beverage Formulation
1. INTRODUCTION
The functional food development that can give solutions for specific requirements
as for sportsmen, diabetics, hypertenses or obese patients, represents a challenge
to food industry, as soon as its legislation and uses (Stephen, 1998).
Undoubtedly proteins are one of the fundamental ingredients in a diet,
since they are the macro nutrients that provide the needed amino acids for the
growth and development of an organism. The demand of protein increases with
the population increase and is necessary a suitable utilization of all existing
sources (Cheftel et al., 1989). In these sense, the bovine plasma which comes
from blood, usually considered a highly contaminant by-product of meat industry,
is an excellent protein source. However, this raw material has disagreeable
sensorial properties, by which it cannot be employed in relatively high
concentrations (del Hoyo et al., 2007). A technological form to improve the
bovine plasma is by means of a concentration-purification process by
microfiltration (MF) and ultrafiltration (UF), followed by discontinues
diafiltration (DD), (Belhocine et al., 1998; Noordman et al., 2002; Cheang and
Zydney, 2004). For proteins concentration, it is necessary to remove impurities,
especially the ions and salts contained in the raw plasma. The prior separation of
these entire compounds from plasma proteins can improve plasma handling (Del
Hoyo, et al., 2007). The removal of the mineral components present in the plasma
is often necessary, if it is to be employed in human foodstuffs, ice creams, cakes,
bread, etc. (Ghost and Cui, 2000, Simon et al., 2002).
In the other hand, a usual method of food preservation by diminution of
water activity is freeze-drying, which simplifies aseptic handling and enhances
stability of dry powders. However, the methods used in proteins processing may
diminish functional properties by loss of protein structures (Toldrà et al., 2008;
Selmane et al., 2008). In this sense, the use of saccharides as stabilizing agent of
protein denaturation has been previously described (Hinrichs, et al., 2001;
Hinrichs, et al., 2005; Buera et al., 2005). Among other saccharides, inulin has
been used as lyoprotector of proteins, its efficiency as protective agent of bovine
plasma proteins has been recently demonstrated (Rodriguez Furlán et al., 2010).
Inulins are a group of naturally occurring polysaccharides produced by many
types of plants. This compound increases calcium absorption, while promoting the
growth of intestinal bacteria (Abrams et al., 2005). Furthermore, it is a soluble
fiber, categorized as a prebiotic (Hempel et al., 2007; Nazzaro et al., 2009). Inulin
has a minimal impact on blood sugar and unlike fructose, is not insulemic and
does not raise triglycerides making it generally considered suitable for diabetics
and potentially helpful in managing blood sugar-related illnesses (Niness, 1999).
In an effort to develop a functional food formulation with combined high
quality proteins and health benefices, the objective of the study were to: i) obtain

International Journal of Food Engineering, Vol. 7 [2011], Iss. 3, Art. 1
a plasma protein concentrate by membrane technology and freeze drying reducing

the protein denaturation by assay the optimal experimental conditions and the use
of a protective agent as inulin; ii) design a beverage formulation sensory
agreeable; iii) examine the use of Stevia as natural sweetness. Stevia, specie
Stevia rebaudiana, was assessed due to it has sweet leaves and many healthy
property in treating obesity and high blood pressure. The vegetal has a negligible
effect on blood glucose and it is attractive as a natural sweetener to people on
carbohydrate-controlled diets. Leaf extract of Stevia rebaudiana promotes effects
on certain physiological systems such as the cardiovascular and renal, influences
hypertension and hyperglycemia. These properties may be correlated with the
presence of antioxidant compounds (Tadhani et al., 2007). Moreover, no evidence
for Stevia constituents causing health risk or birth defects has been found
(Brusick, 2008).
2. MATERIALS AND METHODS
2.1. Raw material
Spray dried bovine plasma (Yerubá S.A. Rep. Argentina) has been used. The
molecular weights of proteins are in the range of 15,000 to 80,000 Da. The
composition is proteins: 76 ± 5, %; ash: 10 %; humidity: 4 % and 1 % of low
molecular weight compounds.
Inulin provided by Orafti Chile S. A., obtained from chicory has been
used. The commercial inulin employed is mainly constituted by linear chains of
fructose, with a glucose terminal unit, and has a molecular weight of 2400 g/mol
(Fig.1 a). This compound is used in a wide variety of foodstuffs as: thickener,
emulsifier, gelling agent, sugar and fat substitutes, humectants, depressing of the
freezing point, among others (Lattanzio et al., 2009).
Stevia in powder (TANKI S. A., Argentina) was used as natural
sweetener. It consists in a mixture of at less eight glycosides diterpenes (Fig.1 b).
For the formulations preparation, some additives as citric acid, Vitamin C,
orange concentrated artificial essence and coloring were used, all of them of
alimentary quality.
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(b)
(a)
Figure 1. Inulin (a) and Stevia (b), chemical structures.
2.2 Analysis
- Determination of protein: The total protein content of the bovine plasma used
as raw material (RM) and the processed bovine plasma (PBP) was determined by
the Kjeldhal method using a Digestion Systems and semi-automatic distillatory
(Selecta, Spain), (AOAC 15017, Association of Official Analytical Chemists,
1990). The conversion factor used to express the results was 6.24. All tests were
carried out in triplicate.
- Ash determination: Samples were weighted into porcelain crucibles and
incinerated in a muffle furnace (Indef, Argentina) with a temperature program to
reach 520 °C (AOAC 15016).
- pH determination: The measurements have been performed with a pHmeter
digital.
- Determination of denatured protein: The soluble protein content was
determined after isoelectric precipitation of denatured/aggregated protein (Meza
et al., 2009; de Wit, 1981, 1990). Solutions of 1% (w/v) protein concentrate were
adjusted to pH 4.8 using 0.1 N of NaOH and HCl. An aliquot of the solution was
centrifuged in a refrigerated ultracentrifuge (Beckman J2-HS, USA) at 20,000
rpm for 30 min at 5 ºC. Protein concentration in the supernatants was determined
by measuring absorption at 280 nm after appropriate dilution in a dissociating
buffer (EDTA 50 mM, urea 8 M, pH= 10) and reported as percentage of the total
protein concentration (Giroux  Britten, 2004). Insoluble protein content of
suspensions at pH 4.8 was defined as the difference between total protein (TP)
and soluble protein (SP) and was used to estimate the extent of denaturation

/aggregation of plasma protein. The percentage of denatured protein (DP) content

was calculated with the following equation (Morr, 1990):
DP 
TP  SP  100 (%)
TP
This determination was performed at every stages of the process to follow

processing effect upon proteins stability.
- Dissolution Time: The time required to dissolve a measured amount of protein

concentrate with and without inulin has been measured. 1 g of the samples was
dissolved in a constant volume (50 ml) of distilled water at 25 ± 2°C, and was
stirred with a mechanical mixer at 1000 rpm. This analysis let to determine the
feasibility that the formulation can be used in instantaneous beverage
preparations.
- Sensory Analysis: A panel of fifty untrained panelists judged the beverages
through the Hedonic scale (Meilgaard et al., 1991). In this affective testing,
samples are presented in succession and the subject is told to decide how much he
likes or dislikes the product and to mark a scale of five points (5 = like extremely;
3= neither like, nor dislike; and 1= dislike extremely). The flavor, aroma, color
and texture were evaluated.
- Statistical analysis: The obtained data were statistically evaluated by the
Turkey-Kramer multiple comparison test in the cases where two or more
comparisons were considered. Otherwise, the T-test was used (SAS, 1989). The
dates from sensory analysis were analyzed by the test of variance ANOVA. A
value of P < 0.05 was considered statistically significant.
2.3. Membrane equipments and procedures
The protein concentration was carried out by means of membrane technology; an

overall schematic representation of the process is shown in Fig.2. In order to
prevent membrane fouling, the feed solutions (3-5 L) were pre-treated by MF
using a frontal flow module (60 m stainless steel filter, Gora). This procedure
reduces the amount of bacteria and spores and acts cold pasteurization. Moreover
the MF stage protects the UF membrane from fouling improving the permeate
flux in UF (Marshall, et al, 1993; Rinaldoni et al., 2009). The UF was performed
by an asymmetric membrane of polyethersulfone located between polypropylene
separators, by a Pellicon cassette system (Millipore, Bedford, MA, USA) with a
molecular weight cut off (MWCO) of 10 kDa, the membrane area was 0.5 m2.
The concentration of proteins by UF was carried out by continuously removing
the permeate stream until the desired concentration of 4 % (w/v), was achieved.
DOI: 10.2202/1556-3758.2014 4

Experimental conditions were established after studies of the variation of

permeate flux as a function of applied pressure and temperature.
A discontinuous diafiltration (DD) process was applied to removal salts
and other contaminant of low molecular weight. This stage can be omitted of the
general process, but the product is less demineralised. For this operation, the
starting material was the UF concentrate, which was diluted to the initial volume
(3-5 L) with treated water in a single state and ultra filtrated to the desired
concentration range (concentration factor of 1.4).
Raw material: Bovine Plasma

Solution: 4% (w/v)
Frontal Microfiltration(MF)
Ultrafiltration (UF) Permeate 1
Diafiltration (DD) Permeate 2
Protein Concentrate
Inulin, 10 % w/v
Freeze-drying
(T: - 40 °C; P: 1 bar; t: 48 hs)
Functional protein powder, for

food formulations preparation
Figure 2. Schematic flow-sheet employed to elaborate the functional protein

powder.

The cleaning of the fouled membrane was performed by applying a

"Cleaning in Place" (CIP) procedure according to the manufacturer's instructions.
At the end of each run, a cycle of water/ NaOH (pH=12.5 ± 0.5)/ water wash was
applied to the membrane at (40  2) °C and at a transmembrane pressure of 1 bar.
Furthermore, a cleaning step using 300 ppm NaClO (commercial grade) was
carried out at the same temperature and pressure to ensure sanitation and cleaning.
Chemical cleaning of the membrane allowed the re-establishing of its
permeability and selectivity in order to maintain optimal performance during the
separation process.
2.4. Freeze drying process
The bovine plasma protein concentrate, obtained by MF-UF-DD was freeze dried
(Fig. 2): i) without protective agent and ii) mixed with a protective agent, inulin at
10% (w/v), which has been demonstrated to be, the most effective concentration
(Rodriguez Furlán et al., 2010). The samples were placed on stain steel trays,
frozen in a freezer at -40 C and freeze-dried using a lyophilizer (Rificor S.A.,
Argentina) at 1 bar of pressure for 48 hours. The samples temperature was
controlled by a temperature sensor. Freeze-drying barely affects the foods texture
and does not provoke extensive retraction or harden their superficial layer.
Moreover, the porous structure makes rehydration a very quick process.
2.5. Beverage formulation design
The recommended daily allowance of proteins, by the World Health Organization

for adult healthy individual is 0.8 g/ per kilo of body weight (Naclerio Ayllón,
2007). In this way, different protein content between 1-5% of the daily
requirement was assayed considering an average body weight of 70 kg. The
freeze dried protein concentrates were used without and with inulin at 10% (w/v).
The amount of Stevia was estimated considering its sweetening power, the rest of
the ingredients were in accord to the legislations for beverages formulation and
were kept constant. Each sample was prepared to be dissolved in a glass of water
(200 ml). The beverage formulations and the function of each ingredient and
additives are showed in Table 1.
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Table 1: Beverage formulation with orange flavor
Ingredients Purpose Amount/ dose Side

1% 2% 3% 4% 5% effects
Bovine Plasma
Concentrate, Amine acid source
0.7g 1.3g 2.0g 2.6g 3.3g None
freeze-dried
without inulin
Amine acid source
Bovine Plasma
Soluble fiber source
Concentrate,
Helps lower blood 2.0g 4.0g 6.0g 8.1g 10.1g None
freeze-dried
cholesterol and glucose
with inulin
levels
Natural sweetness.
Contributes 0 calories
Diminishes the arterial
Stevia
pressure 0.30 – 0.32 g None
Diminishes the appetite
Regulates the glucose
in blood
Flavoring and
Citric acid
neutralizing agent 0.08 – 0.085g None
(acidity regulator)
Vitamin C Preservative, dietary 0.01 - 0.05 g None
(ascorbic acid) supplement
Orange
Flavoring and
artificial 0.30 – 0.32 g None
aromatizing
essence
Carotene,
alpha, beta Coloring 0.5 mg None
and gamma
Beta apo 8 1.3 mg
Coloring None
carotenal

3. RESULTS AND DISCUSSION
3.1 Protein concentration by membrane technology
The frontal microfiltration eliminated insoluble macroscopic components,

reducing the cleaning time of the UF membrane, while the UF-DD steps allowed
concentrating proteins, reducing the saline content.
Preliminary experiments were carried out to evaluate the effects of
transmembrane pressure (P) and temperature on plasma proteins concentration.
The P was varied between 0.5 and 1.7 bar, while the temperature was constant at
10 °C. The result is shown in Fig. 3. The permeate flux increased as the
transmembrane pressure increased, though usually not lineally for
macromolecular feeds. As pressure increases further, the concentration
polarization layer reaches a limiting concentration and the flux becomes
independent of pressure and becomes mass-transfer controlled (Cheryan, 1986).
At his time, the membrane fouling became higher, and so the cleaning more
difficult. From these results it was selected an optimal P of 1.3 bar.
0.006
J (m /m h)
0.004
2
3
0.002
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
P (Bar)
Figure 3. Permeate Flux (J) through the UF membrane, as a function of

transmembrane pressure (T= 10°C).
Increasing the temperature generally increases the permeate flux due to the
dual effect of lowering the permeate viscosity, which assists flow rate, increasing
species diffusivity, (Marshall et al., 1993). However, protein solutions exposed to
mechanical action (as pass through the membrane) are more stable at lower
temperatures, so 10 °C was selected as optimal. With this experimental
temperature, the run time was adequate, while temperatures below this value
made the permeate flux rather slow.
DOI: 10.2202/1556-3758.2014 8

With the operating conditions fixed the DD was performed in order to

deminaralize the plasma concentrates. Fig. 4 shows the ash measurement results.
It can be see that the ultrafiltration followed by diafiltration reduced a 47.5 ±
0.5% of mineral content, obtaining a product apt for food formulation, especially
for diets for e.g. hypertenses or renal patients.
11
10
9
Ash content (%)
4
Raw material MF-UF DD
Figure 4. Ash determination among different stages of the process.
3.2. Protein determination
The total protein in the concentrate without inulin was 84.8 ± 0.8% while for the
protein concentrate with inulin was 27.8 ± 0.4 % (g protein/100 g of product).
From these results, the amount of proteins for the different formulations of Table
1, were calculated.
The diverse treatments applied to proteins produce modifications in their
native conformation due to the hydrogen bonds breaking, preserving intact its
primary structure but exhibiting different effects to secondary, tertiary and
quaternary structures. These changes in molecular structure modify the
nutritional, physical and functional properties (Cheftel et al., 1989). Membrane
processes are mechanical treatments that could modified the protein net,
additionally during freeze-drying the protein is subjected to freezing and drying
stress by which its activity can be lost by conformational change or protein
denaturation (Allison et al., 1999). The results of protein denaturation through the
flow-sheet employed for plasma proteins treatment are showed in Fig. 5.
It can be see that the MF- UF processes carried out with the experimental
conditions selected, effectively reduces the amount of denatured protein. Though
the protein denatured was incremented by the diafiltration process, since it is an

extra mechanical processing, the mineral content was drastically diminished, as

we has previously discussed (Fig.4).
45
Denaturalized protein percentage
40
35
30
25
20
15
10
Raw material MF-UF DD Freeze-dried Freeze-dried
without protect with inulin 10
agent % (w/v)
Figure 5. Protein denaturation among different stages of the process.
During the freeze-dried step, the reduction of denatured protein by inulin

at 10% w/v, was demonstrated. Previous studies have shown that during these
stages, sugars exert two protective effects: i) they replace the water that hydrates
proteins and form with the protein hydrogen-bridge bonds, and ii) the protein is
encapsulated within a vitreous structure avoiding its unfolding and thus
preserving its conformation (Carpenter et al., 1993; Buera et al., 2005). Assuming
that the action mechanism of oligosaccharides is similar to that of
monosaccharides and disaccharides it was proposed that the presence of inulin
displaces and supplants water forming hydrogen bonds with the dry protein which
maintains its structured integrity (Rodriguez Furlán et al., 2010). This result is in
good agreement with the studies carried out by Hinrichs et al. (2001, 2005, 2006)
about inulin as cryo-protectant of protein for pharmaceutical applications.
3.3. Beverage characterization
For a product of instantaneous dissolution, it is necessary to determine the

dissolution time of the matrix. The result for the freeze-dried protein concentrates
obtained by MF-UF-DD without protective agent, was 6.99  0.49 min; whereas
with the use of inulin at 10% w/v the dissolution time was 5  1 s (P < 0.001,
considered extremely significant). The high reduction in the dissolution time was
DOI: 10.2202/1556-3758.2014 10

attributed to the protein spatial conservation by inulin presence. Sugars in solution

act as charges joined upon proteins diminishing protein-protein interactions and
so the macromolecules dissolve easier (Cheftel et al, 1989).
From these protein concentrates, with and without inulin, a beverage
formulation was designed with the ingredients detailed in Table 1. Figure 6 shows
the results of sensory analysis for the different formulations assayed.
A
100
80
Acceptability %
60
40
20
Color
Aroma
0
Flavor
1% Texture
2% 3%
4%
Daily pro 5%
tein requ
ire ment
B
100
80
Acceptability %
60
40
20
Color
Aroma
0 Flavor
1% 2% Texture
3% 4%
Daily pro 5%
tein requ
irement
Figure 6. Sensory Analysis of beverage formulations with different protein

content: A: Bovine Plasma Concentrate, without inulin; B: Bovine Plasma
Concentrate, with inulin (10% w /v).
11

The formulation with inulin had greater acceptability by the panel, for all
attributes evaluated. The presence of inulin softened the flavor and color of the
beverage. The formulation without inulin showed an average acceptability of 48.2
± 8.5 (P <0.001 considered extremely significant). This result is related with the
high dissolution time and the formation of lumps, getting a low score in texture.
However, both products improved with the removal of compounds that provide
poor color, taste and smell during the stage of MF-UF-DD. It was determined that
the formulation with 3% of the protein coverage requirement (for an average body
mass of 70 kg) presented a high acceptability (attributes analyzed average of 86.5
± 0.8), with higher protein content, the graph of acceptability declined in average
13%.
Therefore, the beverage formulation obtained was an orange powder with
agreeable flavor, color, aroma and texture, which was accepted by approximately
the 90% of the judge’s panel. It was feasibly dissolve in water. The pH value of
the beverage was 4.8 ± 0.1. The product is stable and in a solid state can be easily
preserved from damage. The formulation has high quality proteins without
phosphorous and sugars, with low saline content additionally ingredients that
confer functional characteristic, are present. It is important to remark that, Stevia
effect as glucose regulator in blood and arterial pressure reducer, makes it would
be helpful in the management of renal deficiency, diabetic, hypertenses patients or
low-calorie diets. Normal digestion does not break inulin down into
monosaccharides and so it does not elevate blood sugar levels, moreover the
soluble fiber reduces cancer risk. More studies may be necessary to assay the use
of this beverage in each case.
4. CONCLUSIONS
The functional properties of dietary proteins are important in food processing and
formulation of nutritional products, since they contribute to the desired
characteristics and quality that the consumer perceives. Besides, the current
tendency towards nutritional formulation using refined ingredients results in a
greater demand of food showing high-quality protein content (Chove et al., 2007;
Yu et al, 2007).
The formulation of a beverage with nutritional and functional properties
was analyzed. The bovine plasma concentrated and demineralised with membrane
technology, was used as protein source. The protein concentrate was freeze-dried
with and without inulin as protective agent. The addition of inulin demonstrated
that reduces the protein denaturation and so functional properties are better
preserved, allowing application for food formulations with proteins. Furthermore,
the porous powder obtained has functional properties by the inulin presence and it
DOI: 10.2202/1556-3758.2014 12

has a high solubility with high acceptance in the sensory analysis. The results let
to give higher added value to proteins obtained from blood, traditionally
considered as a meat industry residue. The likely cost of production of the
beverage would be similar to other protein preparations, taking into account the
raw materials and the processes employed. For all these reason, it is important to
highlight that the combination of the product with Stevia and other additives, let
to arrive to a beverage with exceptional qualities that can be applied as protein
supplement for different purposes.
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DOI: 10.2202/1556-3758.2014 16


Development of A Functional Beverage

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Development of A Functional Beverage

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International Journal of Food

Development of a Functional Beverage

Laura T. Rodriguez Furlán, INTEQUI-CONICET-UNSL

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KEYWORDS: plasma protein, ultrafiltration, functional beverage, inulin, Stevia.

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a plasma protein concentrate by membrane technology and freeze drying reducing

2. MATERIALS AND METHODS

2.1. Raw material

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Figure 1. Inulin (a) and Stevia (b), chemical structures.

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/aggregation of plasma protein. The percentage of denatured protein (DP) content

This determination was performed at every stages of the process to follow

- Dissolution Time: The time required to dissolve a measured amount of protein

2.3. Membrane equipments and procedures

The protein concentration was carried out by means of membrane technology; an

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Experimental conditions were established after studies of the variation of

Raw material: Bovine Plasma

Ultrafiltration (UF) Permeate 1

Diafiltration (DD) Permeate 2

Functional protein powder, for

Figure 2. Schematic flow-sheet employed to elaborate the functional protein

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The cleaning of the fouled membrane was performed by applying a

2.4. Freeze drying process

2.5. Beverage formulation design

The recommended daily allowance of proteins, by the World Health Organization

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Table 1: Beverage formulation with orange flavor

Ingredients Purpose Amount/ dose Side

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3. RESULTS AND DISCUSSION

3.1 Protein concentration by membrane technology

The frontal microfiltration eliminated insoluble macroscopic components,

Figure 3. Permeate Flux (J) through the UF membrane, as a function of

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With the operating conditions fixed the DD was performed in order to

Figure 4. Ash determination among different stages of the process.

3.2. Protein determination

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extra mechanical processing, the mineral content was drastically diminished, as

Figure 5. Protein denaturation among different stages of the process.

During the freeze-dried step, the reduction of denatured protein by inulin

3.3. Beverage characterization

For a product of instantaneous dissolution, it is necessary to determine the

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attributed to the protein spatial conservation by inulin presence. Sugars in solution

Figure 6. Sensory Analysis of beverage formulations with different protein

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Buera, P., Schebor, C. and Elizalde, B., Effects of carbohydrate crystallization on

Cheang, B. and Zydney, A. L., A two-stage ultrafiltration process for fabrication

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Cheryan, M. Ultrafiltration. Handbook.,1986, Technomic Publishing Co., INC,

Chove, B. E., Grandison, A. S. and Lewis, M. J., Some functional properties of

de Wit, J.N., Structure and functional behaviour of whey proteins, Netherlands

de Wit, J.N., Thermal stability and functionality of whey proteins, Journal of

Ghost, R. and Cui, Z. F., Protein purification by ultrafiltration with pre-treated

Hinrichs, W. L. J., Sanders, N. N., De Smedt, S. C., Demeester, J. and Frijlink, H.

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