UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ - NAPOCA

DOCTORAL SCHOOL

FACULTY OF ANIMAL SCIENCE AND BIOTECHNOLOGIES

ENG. IANCU RAMONA MARIA

UNCONVENTIONAL TECHNOLOGIES, COMPLYING WITH EUROPEAN REGULATIONS, FOR IMPROVING

THE NUTRITIONAL AND HYGIENIC QUALITY OF GOAT MILK

SUMMARY OF PhD THESIS

SCIENTIFIC COORDINATOR

PROF. PhD LETIŢIA OPREAN

Cluj - Napoca

2011

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CUPRINS  

 

INTRODUCTION. PURPOSE AND OBJECTIVES ……………………............ 3 CHAPTER 1  ………………………………………………………………………………………………. 6 IMPORTANCE AND EVOLUTION OF GOATS’ GROWTH  1.1. Social and economic importance of goats’ growth …………………………………. 6 1.2. Goat sector development  ………………………………………………………………………. 7 1.3. Goats’ growth outlook in our country ……………………………………………………. 8 CHAPTER 2 …………………………………………………………………………………………….. 8 GOATS’ NUTRITION AND FEEDING BASICS  2.1. Forage resources and their nutritional assessment …………………………………… 8 

2.1.1. The importance of fodder and its classification  ……………………….. 9  2.1.2. The role and use of food in the body  ……………………………………….. 9  2.1.3. Notions of fodder and nutritional value. Estimation of fodder                   nutritional value ………………………………………………………………………… 10

2.2. Goats nourishment …………………………………………………………………………………. 11CHAPTER 3 …………………………………………………………………………………………… 11NUTRITIONAL AND HYGIENIC QUALITY FEATURES OF GOAT MILK  CHAPTER 4 ……………………………………………………………………………………………  13EXPERIMENTAL RESEARCH ON IMPROVING THE MICROBIOLOGICAL QUALITY OF GOAT MILK  4.1. Materials and methods …………………………………………………………………………… 134.2. Results and Discussion  ………………………………………………………………………….. 144.3. Conclusions ……………………………………………………………………………………………. 16CHAPTER 5 …………………………………………………………………………………………… 16EXPERIMENTAL RESEARCH ON THE NUTRITIONAL QUALITY OF GOAT MILK  5.1. Materials and methods ……………………………………………………………………………. 16 5.2. Results and Discussion  …………………………………………………………………………. 175.3. Conclusions …………………………………………………………………………………………… 24 CHAPTER 6 ……………………………………………………………………………………………. 25RESEARCH ON IMPROVING THE NUTRITIONAL QUALITY OF GOAT MILK BY USING VITAMIN B12 SUPPLEMENTS  6.1. Materials and methods ……………………………………………………………………………. 256.2. Results and Discussion …………………………………………………………………………… 276.3. Conclusions  …………………………………………………………………………………………… 33GENERAL CONCLUSIONS …………………………………………………………………… 34REFERENCES ……………………………………………………………………………………… 37

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INTRODUCTION: PURPOSE AND OBJECTIVES

Goat milk is the natural food product which provides the body all the necessary

nutrients for growth and development. The consumption of one liter of milk fills up the

needs of a grown-up man regarding fats, calcium and phosphorus, 53% of the RDA

(Recommended Dietary Allowance) of protein, 35% of the RDA of vitamins A, C, B and 26%

of the energy needs.

Milk vitamins are non-protein origin substances, of a major biological importance,

which catalyze the metabolic processes of living organism. The goat milk deficiency, in

comparison with the cow milk it is also determined by the vitamin B12, which is found in

an amount up to four times lower in goat milk than in the cow milk.

The aim of this thesis was to evaluate the physical-chemical and

microbiological composition of goat milk and development of non-conventional

methods to improve the nutritional and hygienic quality of raw goat milk.

The study objectives were:

1. Determining the quality of goat milk produced by different production systems on

the influence of the specific pathogen agents from milk: staphylococcus,

streptococcus and Escherichia coli.

2. Determining the relationship between the total number of somatic cells and the

udder infection in different production systems.

3. Evaluation of the procedures used to obtain milk in good hygienic conditions, in

different production systems.

4. Regular checks of milk production at the goats in two different geographical areas.

5. Quality measurement of goat milk according to the season and lactation stage.

6. Determination of vitamin B12 in animal forage.

7. Research on the content of vitamin B12 in various food supplements.

8. Development of non-conventional biotechnological methods to improve the

nutritive quality of goat milk, using supplements of vitamin B12.

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9. Appreciation of the nutritive quality of goat milk enhanced with vitamin B12

supplements.

Structure of the thesis: It is divided into two parts, the literature study and original

contributions.

Part I covers the literature study and includes three chapters:

Chapter I – presents the social and economic importance of goats’ growth,

dynamics of the goat sector development and its growth perspective in our country.

Chapter II - shows the importance, structure and assessment of nutritional forage

basis and the role of goat nourishment in milk production.

Chapter III – presents the nutritional and hygienic quality characteristics of goat

milk, describing the physical and chemical properties of goat milk and its chemical

composition.

Part II covers the research made and original contributions divided into three

chapters:

Chapter IV - presents the experimental research on improving the microbiological

quality of goat milk, by the analysis of conformation and health of the goat udder, by

determining the hygiene degree of milk and water.

Chapter V - presents experimental research on the nutritional quality of goat milk

by monitoring the physic-chemical index in the two farms, according to season and

lactation stage.

Chapter VI - presents the research on improving the nutritional quality of goat

milk using vitamin B12 supplements, from the determination of vitamin B12 in animal

forage and other food supplements.

The structure of the thesis is dedicated to the full achievement of the proposed

objectives, with six chapters, extended on 224 pages, including 35 tables, 43 figures and a

bibliographical list of 178 representative titles, significant in the presentation and

description of the goats’ growth.

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The novelty of the thesis consists of a different approach regarding the

requirements of a much healthier and safer food, growing concern for human health, its

relationship with the structure and quality of food, as well as the association of some

micronutrients, especially vitamins, with beneficial effects upon health. Goat milk

enhanced with vitamin B12 supplements are a necessary food product for the elderly.

When the digestive system is unable to absorb this vitamin and an inadequate diet

typically for confident vegetarians, who do not consume any milk products or eggs, it can

generate a lack in vitamin B12 and a deficiency occurs, which produces: palpitations,

weakness, loss of appetite and memory, personality disorder.

Scientific studies conducted over a period of three years are the result of the

collaboration between the Research Centre in Biotechnology and Microbiology of the

University “Lucian Blaga” of Sibiu and the National Research Institute for Biological

Sciences, Bucharest.

The thesis is the result of the author’s research, supported and encouraged by

people who understood the need to ensure a rational diet, well balanced in nutrients.

I sincerely and respectfully address my thanks and gratitude to Mrs. Prof. PhD Letiţia Oprean, as a scientific leader of this PhD thesis, for the relevant guidelines and

recommendations made during the entire period of performing the work. I also thank

Mrs. Professor for the support, encouragement and the confidence given.

Sincere thanks to the scientific reviewers’ gentlemen, for their patience to analyze

the PhD thesis, for the feedback, suggestions and comments brought to the content of the

paper.

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CHAPTER 1

IMPORTANCE AND EVOLUTION OF GOATS’ GROWTH

1.1. SOCIAL AND ECONOMIC IMPORTANCE OF GOATS’ GROWTH

With the current rapid growth of world population and prolongation of human life,

the raise of the living level and the targeting of food to a growing extent of agro-food

products, with high nutritional and biological value, the need for food, especially of

animal origin have increased ever more.

Livestock, as the main branch of the national economy it is important in that it

provides products that have different uses and by contributing to the increase of the

profitability of agriculture as a whole (DAVIDESCU, 1994; DAVIDOVICI, 2002,

GEORGESCU, 2003). Thus, from animals there are obtained, through the process of

growth, food products which can be replaced only partially by foods of plant origin

(BAHCIVANGI, 1999; OPREAN, 2003).

Food products used in human diets are superior to those of plant origin by: traits

taste, high nutritional and biological value and high digestibility. Because of these

characteristics, animal foods are indispensable for rational nutrition of the people,

especially children, the sick, convalescent and the elderly.

Goats capitalize the crops obtained on natural pastures and natural mountain

meadows, areas that cannot be used for cereal crops and can exploit a number of major

industrial waste from large enterprises like bakery business, manufacturers of alcohol,

beer, starch, vegetable oils and sugar, such as: bran, sharps, cakes and flower bran, soy,

marcs, molasses and sugar beet noodles.

The use of natural fertilizers produced by goats helps to improve soil fertility and

thereby increase crop production.

Goats’ rearing allows a more efficient use of agricultural employment in the

existing establishments. Unlike the plant sector, characterized by peaks of productive

activity, in the livestock sector work is carried out continuously throughout the year.

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Thanks to the used technologies, there may be offered jobs for a large part of the

active population in the country and can be obtained a significant money income

throughout the year by the superior capitalizing of animal products. Goats’ rearing is one

of the agricultural branches that provide maximum opportunities to enhance the

production process, the industrialization of technologies, of growth and operating.

1.2. GOAT SECTOR DEVELOPMENT DYNAMICS

Livestock is one of the oldest human pursuits. It began with the domestication of

animals and has continuously developed, alongside the evolution of human society. Each

stage of development of human society corresponds to a certain level of breeding.

The level of development of goat growth is largely determined by the performance

of scientific research system, of science generally speaking. Highly skilled human

resource, creativity, production and using of knowledge have proven to be, during time,

decisive factors in the economic development of highly developed countries.

The transition to a knowledge-based society is a fundamental strategic option and

has a great impact on the growth of goats, the agricultural research units having a vital

role through contributing to the production, transmission, dissemination and use of

knowledge.

At the end of the year 2008, the total number of sheep and goats is increasing by

4.8% and the number of females (sheep and goats) increased by 5.3%. In terms of

animals per 100 ha, our country is placed after: Greece, United Kingdom, Portugal,

Spain, Holland, Italy and Ireland (INSS, 2007).

Another important issue affecting the development of goat industry in our country,

is it also the increase and improvement of the grazing areas. Of course, creating a

network for leather, meat and goat milk in Romania, based on the structure of goat

associations or other associations, as well as providing marketing for the education of

people to consume milk and goat meat, may positively influence the goat sector.

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1.3. GOATS’ GROWTH PERSPECTIVE / OUTLOOK IN OUR COUNTRY

The integration of our country in the EU has been a strategic option shared by the

entire spectrum of the political forces in Romania. For the Romanian farmers, the

achievement of this goal meant the opening of a vast market of food products,

characterized by a high potential for absorption, high purchasing power and relative

stability of prices.

At the same time it must be taken into account that on this single market (of which

an integrated part will become “the internal market” from Romania) the competition is

and will be extremely tough, in terms of price and quality of supply and promotion

policies of products.

Prosperity or failure of different farmers in this market is determined by the level

of competitiveness, in turn ensured by the economic performances of the involved

“actors” and the economic environment in which they act (TAFTĂ, 2002).

Application of modern improvement systems on goats involves supplementing

traditional methods used to improve the genetic potential with methods for assessing the

hereditary basis and their productive capacity so that decisions can be taken fast and

efficient during the selection and breeding process.

CHAPTER 2

GOATS’ NUTRITION AND FEEDING BASICS

2.1. FORAGE RESOURCES AND THEIR NUTRITIONAL ASSESSMENT

The knowledge of food role on all animal body functions and of the rational

nutrition principle outlines the means of achieving the increased production of forage,

according to the requirements of livestock development.

Through a rational diet it is provided a complete food, in terms of nutrient content,

depending on the specific morph-productive and physiological state of animals at a time.

Always a reasonable diet, well balanced in nutrients ensures good maintenance and high

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animal production (ZAMFIRESCU, ŞONEA, 2004, IANCU, 2010). Nutrition directly

affects, not only the level of animal production, but also the normal reproductive

function, the processes of growth and development, animal health and economic

efficiency.

In the study of forage and the technique of feeding the animals, the most important

principle is the importance of the relationship between forage characteristics and their

digestive process.

Fodder with good taste qualities increases the appetite of animals, which results in

an increased activity of digestive gland secretion, a more complete digestion and better

assimilation of nutrients.

2.1.1. The importance of fodder and its classification

The problem of goats’ nourishment, through the level and quality of forage given

during the calves’ period and the grazing period in the warm season, is one of the factors

that are under the control of the breeder and which may ultimately decide the economic

profitability and operating goats’ growth.

Stimulation of feeding and with good quality forage, in the necessary quantities of

various physiological states of the goat, is the prerequisite to obtain high production,

good health and some raised indices regarding reproduction (PĂDEANU, 2000).

Fodder used to feed goats can be of plant, animal or mineral origin. Plant fodders

are obtained in the process of agricultural productions and from the technical processing

industries of the plant raw material. Animal fodder is obtained from slaughterhouses, the

canning industries and from natural resources. Mineral fodder is made from mineral

rocks processing and from the chemical industry. All feeding resources, together, form

the basis of forage.

2.1.2. The role and use of food in the body

Nutrients are used in the body to produce the necessary energy for animals and for

material synthesis for the growth of tissues or tissue repair worn by functioning, at the

synthesis of sex cells, hormones, vitamins, for the most part, of nutritive substances that

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are used to prepare the products we get from animals. Therefore, food has an energetic

and a plastic role in the body. The main source of energy for the body consists in

carbohydrates and fats. Carbohydrates and fats are oxidized to carbon dioxide and water

and proteins are metabolized to the final stage of carbon dioxide, water and ammonia, in

both cases with the release of energy. The plastic role of food is to provide the necessary

substances for tissue growth in young animals, to repair the adult used tissue in mature

animals, for milk production.

2.1.3. Notions of fodder and nutritional value

Estimation of fodder nutritional value

The criteria for assessing the nutritional value of forage and their interpretation

have been successive and completed in time, using, in turn, different criteria:

- the content in some groups of nutrients considered as being rough: it is a method

to assess the nutritional value of forage after the total gross of substances, after the

nitrogen content of substances, after the non-nitrogen content of substances; provides first

hand information on the nutrient composition of forages .

- the content of digestible nutrients: expressed by the total digestive organic

nutrients, caloric equivalent (T.S.D.), determined by chemical analysis and digestibility

experiments;

- the energetic value, interpreted by: the action of forage energy on the growth of

the body energy and the energetic value represented by the digestible or metabolized

energy of the fodder;

- appreciation of forage only on the basis of energy is yet unilateral, because it is

completed with data which indicates, besides the energetic value, also the content of

substances with plastic and functional role, important to the life and animal production,

such as: proteins, vitamins and mineral salts.

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2.2. GOATS NOURISHMENT

The proper nutrition of goats is important at all ages (HETHERINGTON, 1994).

Nutritional requirements differ in certain stages of maintenance, growth, gestation and

lactation and those refer to the insurance of the right level of energy, protein, minerals,

vitamins and water (HALGA, 2005). Being a ruminant animal, the goat has, from the

nutritional point of view, the possibility to storage in a voluminous digestive system some

fibrous vegetable forage with high content of cellulose, unusable by the mono-gastric

animal species. The goats’ nourishment in the Romanian farms is provided by grazing,

from May until October or November, the period of calves being more than 5 months.

For this reason, the stored quantities of hay are moderate (2006 - 142 kg/year/goat; 2007 -

149.78 kg/year/goat). In 2006, the distribution of concentrates in farms is high (0.534

kg/l) for an average production of 224.23 l /goat. The supplement of concentrates

decreased in 2007 (0.325 kg/l), in terms of a higher production of milk, of 281.80 l/goat.

The amount of mineral supplements provided in the diet of goats was

11.7 kg/goat /year in 2006 and very low for the year 2007 (3.57 kg/goat/year). For the

Carpatina breed, the milk production over a lactation period of 180-210 days is low, in

average it is 224.23 l/goat in 2006 and 281.80 l/goat in 2007 (ZAMFIRESCU, 2009).

CHAPTER 3

NUTRITIONAL AND HYGIENIC QUALITY FEATURES OF GOAT MILK

The application of modern breeding systems of goats involves supplementing the

traditional methods used to improve the genetic potential with methods for assessing the

hereditary basis and their productive capacity so that decisions can be made faster and

efficient in the selection and breeding process.

Goat milk is considered superior to the cow milk because of its nutritive, tonic and

anti-rachitic, anti-anemic, and anti-infective effects. It has specific, sweet taste and smell,

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in case of hygienic maintenance and milking and proper feeding, especially in terms of

fodder varieties (TAFTĂ, 1996).

Due to the fineness of fat cells, goat milk is easily absorbed by intestinal villi and

thus has a higher digestibility. Under the action of gastric juice proteins coalesce into fine

flakes, are easily digested by proteolyses enzymes of the small intestine and, therefore,

are easily assimilated. Goat milk, being richer in calcium, phosphorus, citric acid,

potassium and magnesium and having a high content of vitamin A is indicated in the diet

of infants, of the elderly (TIŢA, 2002). Since goats are very rarely ill of tuberculosis, goat

milk can be consumed in raw state, thereby retaining the maximum efficacy of the

proteins, lipids, sugars, vitamins, enzymes and mineral salts (TAFTĂ, 1996).

The chemical composition of goat milk is the subject of race, individuality, area,

age, level and type of food, stage of lactation, season calendar, milking duration and

health status (ANTUNAC, 2001). The results obtained in numerous studies have shown

that regarding the composition, goat milk is generally much alike to cow milk.

In a review conducted by BOUDIER et al., 1981 and JOUZIER et al., 1986, are

presented the main components of milk in domestic ruminant species (Table 1)

(JOUZIER, COHEN-MAUREL, 1986).

Table 1

The chemical composition of milk

Specification Measuring unit Cow Buffalo Goat Sheep

Energy Kcal/l 650-720 755-1425 600-700 1050-1150 SU % 12,6-12,8 16,6-17,5 11,3-13,4 17,5-18,3 Fats % 3,7-3,9 6,8-7,9 3,3-4,1 6,6-7,1 Proteins % 3,3-3,4 4,0-4,1 2,9-3,3 5,2-5,7 Lactose % 4,7-4,8 4,8-5,0 4,4-4,8 4,6-4,9 Mineral salts % 0,7-0,9 0,7-0,8 0,7-0,8 0,8-0,9

 Source: Jouzier, Cohen‐Maurel, 1986 

The data used by authors show that for both the energy value and content in

protein, fat, lactose and mineral salts, goat milk is similar to cow milk values.

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CHAPTER 4

EXPERIMENTAL RESEARCH ON IMPROVING THE

MICROBIOLOGICAL QUALITY OF GOAT MILK

4.1. MATERIALS AND METHODS

To obtain information about the production of goat milk and goat rearing for milk

by farmers, it was used an analysis questionnaire. In the study area, through the

questionnaire, there were selected two farmers, located in the county of Sibiu and Vâlcea.

These farms were regarded as representative for two different systems of production and

were classified as follows: FLMS Farm, in Sibiu County, with extensive production

system and FLA Farm in Vâlcea County with semi-intensive production system. In the

two farms prevails the Carpatina race and less the White Banat. From each farm, there

were collected milk samples from 15 goats, from each udder. A total number of 270 milk

samples from half the udders have been analyzed from the two commercial flocks studied

for qualitative analysis, observation of the milk collection process and study of factors

contributing to milk hygiene.

Conformation and udder health. Udder examination was made to determine the

variation in the udder and nipple conformation by observing the following indicators:

symmetry and udder attachment, udder shape and its opening.

Milk and water hygiene. Qualitative and quantitative analysis of pathogens,

samples of milk and water, was performed to determine the milk hygiene, as follows:

1. Analysis of milk sample. The analysis of milk samples taken from the half udder

was done by determining the probable number of germs (TBC). The method consists in

determining the organic- trophic aerobic bacteria by sowing in Petri plates on agar blood

tryptose medium. The microbial cells present in the sample in contact with agar nutrient

medium will each form visible colonies after incubation at 300C for 72 hours. Taking into

account the dilution used and the number of developed colonies, it is determined the

number of bacteria per 1 ml. (CFU/ml). There were also applied the potassium hydroxide

test to differentiate Gram positive and Gram negative bacteria, catalase test to highlight

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the presence or absence of staphylococcus and phosphate test to highlight the difference

between Staphylococcus aureus and other staphylococcus that do not secrete the enzyme

called coagulase, by testing the presence or absence of the clotting enzyme.

2. Analysis of the homogeneous sample of milk: The Petrifilm method can be used

to determine the total number of mesophilic aerobic germs, the coliform bacteria and

Escherichia coli bacteria in milk, dairy products, but also from the surfaces of the

equipment.

3. Water sample analysis: From each farm it was taken a sample of water from the

existing source, at each visit. This has been tested to determine hardness and

microbiological quality (number of standard colonies, coliform bacteria and Escherichia

coli).

4.2. RESULTS AND DISCUSSION

Conformation and udder health. The analysis revealed the following:

The goats under the extensive production system, had presented a better udder

conformation than those raised under the semi-intensive production systems. To note that

there were very few animals with teat orifice on a larger scale of 1 (7%). Strong udder

attachment was observed at the goats under semi-intensive growth system, with 80% of

teats in scale of 1. It may be inferred from the number of lactations and parity that most

of these animals were mature. Coagulase - negative staphylococcus (CNS) were the most

common microorganisms, responsible for 85.71% of the half infected udders. They

included Staphylococcus epidermidis and Staphylococcus intermedius. Staphylococcus

aureus was another pathogenic microorganism identified in the udders of goats as a

percentage of 14.29% (OPREAN et al., 2009, 2010).

Milk and water hygiene. The hygiene of milk varied in different production

systems. The total number of bacteria in raw milk, showed a large contamination of milk

produced in the extensive production system. The average number of TBC was 36 300

CFU/ml in extensive system and 16 450 CFU/ml in semi-intensive system. According to

the International Federation of Animal Growth Farms for Milk, the milk with TBC values

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less than 20 000 CFU/ml, reflects good conditions of hygiene, such as the semi-intensive

production system. In the case of the extensive system, coliform bacteria were most

common in the homogeneous sample of milk. The largest number of coliform bacteria

was found in this system, with 22 coliform bacteria/ml of milk, in comparison with the

semi-intensive system (7 coliform bacteria / ml).

Most coliform bacteria are obtained from water, as other authors have sustained

previously, this variation being attributed to water quality from wells (GILMOUR,

ROWE, 1981, SANDHOLM et al., 1995b).

This idea is maintained by the bacteriological quality of water used in the

extensive production system, which showed a TBC count of 523 CFU/ml (standard law:

100 CFU/ml, according to European Regulation 853/2004 and 854/2004). This shows

that the farm, like most small farmers, rely on the untreated water supplies from wells.

Some of these may be contaminated at source, through a variety of saprophytic

organisms, from soil or vegetation.

Despite the fact that the used water in the semi-intensive system came from a well

and had a TBC count of 85 CFU/ml, there was a lower contamination with coliform

bacteria. This could be due to the way in which the udder was ready for milking, being

wiped with a disposable paper towel. The installation of milking to the container was also

rinsed with a disinfectant. This explains the number of coliform bacteria in the normal

parameters (less than 20 NBC/ml). Cases of bacterial contamination, in the semi-

intensive system, can be attributed to intra-mammary infections.

Based on the obtained results, the correlation value of Spearman was 0.421,

showing a correlation between increasing parity and the produced quantity of milk

(p ≥ 0.00621).

Determining of the number of somatic cells was also performed for a

homogeneous sample of milk. Under the extensive production system, SCC was of

376,000 and for semi-intensive system were 197,000, both values being below the

standard value of 400,000 somatic cells/ml milk. On this basis, the correlation between

somatic cell counts equal to or greater than 400,000 cells /ml of milk and the presence of

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infection in goat milk, was determined by using the Fisher test, for association (p = 0.2)

in the different production systems (ARDELEAN, 2006).

4.3. CONCLUSIONS

1. The coagulase- negative staphylococcus (CNS) were the most common

organisms (Staphylococcus epidermidis, Staphylococcus intermedius and Staphylococcus

aureus), responsible for 85.71% of the half infected udders (udder inflammation =

mastitis). Staphylococcus aureus was identified in the goat’s udders at the rate of 14.29%.

2. In the studying period of the early lactation, it has been found that only by

relying just on somatic cells, one cannot predict the health of the goat’s udder. Much

more is guaranteed the isolation of bacteria in milk samples directly from the udder.

3. The study shows that the extensive production system is more favorable for

facilitating small farms of goats’ growth because it doesn’t require drastic changes in

infrastructure, it is not labor- intensive and the farmer has complete control upon the

activity. Hygiene can be enhanced by the extension services regarding the clean milking

procedures and sampling of pathogens (Staphylococcus aureus, Enterococus faecalis) can

be minimized by a microbiological strict control program.

CHAPTER 5

EXPERIMENTAL RESEARCH ON NUTRITIONAL QUALITY OF GOAT MILK

5.1. MATERIALS AND METHODS

In the selected farms for the determination of physic-chemical composition of

milk, are predominate two breeds of goats, Carpatina breed and White Banat breed, both

distinguished between them by productivity, as well as by their training module and their

geographical distribution area.

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To perform the physic- chemical analysis, there were collected a total number of

15 samples/ month from each farm throughout the four seasons: spring, summer, autumn

and winter.

A total number of 270 samples were analyzed using Ekomilk Total milk analyzer

located in the equipment of the Center for Research in Biotechnology and Microbiology

of the University „Lucian Blaga” of Sibiu. Sampling was done in sterile vials of 50 ml,

which were then labeled and placed in a cold box at a temperature of 4°C to the

laboratory, where they were analyzed. The sampling procedure was accompanied by an

individual file which included the identification number of the goat, lactation number,

date of harvest, the amount harvested and the physic-chemical indices analyzed: fat,

protein, SNF, lactose, pH, density, conductivity and freezing point. Measurements were

made according to season and stage of lactation.

5.2. RESULTS AND DISCUSSIONS

The composition of goat milk regarding the major and minor components depends

on the season, but also on the winter feeding (silage, hay, concentrates).

Table 2

Variability of physical-chemical index and average values of goat milk depending on the

season, at the farm FLMS, Sibiu district

Analysis index Period Fat SNF* Density Protein Lactose pH

Spring n=60 x±sx 4,89±0,03 9,52±0,03 1,03±0,01 4,13±0,04 4,36±0,02 6,31±0,02

Summer n=60 x±sx 3,95±0,07 8,95±0,05 1,029±0,01 3,78±0,02 4,39±0,03 6,71±0,02

Autumn n=60 x±sx 4,95±0,06 9,22±0,03 1,03±0,01 3,96±0,02 4,42±0,02 6,50±0,02

Winter n=60 x±sx 5,21±0,04 9,64±0,03 1,033±0,01 4,42±0,03 4,30±0,02 6,32±0,02

* Solids non fat content

The variability of the physic-chemical parameters and their medium values shows

differences. Following the research carried out, the highest average value obtained for

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fats is recorded in the winter with 5.21% ± 0.04. The highest content of fats recorded

during winter is due to the introduction of good quality alfalfa hay and of oat bran in the

animal food. Unlike the content of green mass - 200 g D.M. /kg fodder, the amount found

in alfalfa hay and oat bran is much higher, of 900 g D.M. /kg fodder (IANCU, 2010).

The maximum average value of non-fat dry matter obtained from the tests carried

out exceeds the amount determined by CHINŢESCU and THOMA in 1999 (8.9%), this

being in the winter of 9.64% ± 0.03.

The content of protein, like the one in fat has the highest average value also in the

winter of 4.42 ± 0.03, due to different feeding during winter.

The density of goat milk in the four seasons was maintained at around 1,029±0, 01

and at 1,033±0, 01.

The lactose production is important, as long as it regulates the release of water by

the mammary tissue. At the same time, the quantity of milk is strongly correlated to the

level of lactose. The concentration of lactose in milk is rather constant, reaching overall

values of 4.4%. In the figure 1 are represented the main physic-chemical indices

determined at the goat milk depending on the season at the farm FLMS, Sibiu county.

Fig.1.Variability of physical-chemical index and average values of goat milk, depending

on the season, at the farm FLMS, Sibiu county.

XVIII  

Table 3

Variability of physical-chemical index and the average value in goat milk, depending on

the season, at the farm FLH, Vâlcea County

Analysis index Period Fat SNF Density Protein Lactose pH

Spring n=60 x±sx 5,10±0,05 9,31±0,03 1,03±0,01 4,45±0,02 4,37±0,02 6,44±0,02

Summer n=60 x±sx 4,20±0,07 9,12±0,04 1,03±0,01 4,22±0,02 4,20±0,03 6,75±0,02

Autumn n=60 x±sx 4,70±0,04 9,27±0,05 1,031±0,01 4,32±0,03 4,40±0,02 6,35±0,02Winter n=60 x±sx 5,85±0,03 9,72±0,03 1,031±0,01 4,68±0,03 4,51±0,03 6,32±0,02

Fat is one of the most important components of goat milk regarding the

organoleptic properties, but also from its nutritional and economic point of view with

maximum values during the winter 5.85 ± 0.03 (IANCU, 2010). The lactose gives the

sweet taste of milk. Lactic bacteria convert lactose into lactic acid, which causes milk to

sour (increasing acidity). The conversion of lactose into lactic acid is called lactic

fermentation, a particularly important phenomenon in the production of fermented milk

products and cheese. Regarding the content of lactose, the highest average value of 4.51

± 0.03 is found in winter and the lowest in summer.

0

1

2

3

4

5

6

7

8

9

10

Valori m

edii ale indicilor fizico‐

chim

ici/Th

e average conten

t of 

physical and

 che

mical indices

Primăvara/Spring Vară/Summer Toamnă/Autumn Iarnă/Winter

Sezon/Season

Grăsime/Fat

SNF

Densitate/Density

Proteină/Protein

Lactoză/Lactose

pH

Fig. 2 Variability of physical-chemical index and the average values of goat milk,

depending on the season, at the farm FLH, Vâlcea County

XIX  

To cover the various nutrients in goats, energy and protein supplied by food

should be increased, as these milk animals have a smaller rumen capacity. Green fodders,

including silage, rich in fiber and/or water are poor in energy and protein components. On

the other hand, grain, fat seeds (sunflower, soybeans) contribute to the increase of the

intake energy of goats and the flours from defatted oilseeds increase the amount of the

protein in food. Goats, as well as the sheep, require daily an amount of fiber in their diet,

to prevent the installation of the acidosis state in the rumen, state which is leading to

parakeratosis and enterotoxaemia that would help to decrease fat content in milk and

possibly, increase the protein content. A diet with proper proportion of hay, silage and

concentrates as mixed or as pellets or cubes will usually lead to more milk production,

changes in milk fat and protein content, changes that will affect the manufacture of

cheese. At the same species and breed, lactation period has a strong influence on the

composition of milk. As for goats, both colostrum milk and that from the first period of

lactation is richer in fat and protein, then these components decrease and then increase at

the end of the lactation period, when the milk production is lower.

Table 4

The variability of physical-chemical index and average values of goat milk, depending on the lactation stage at the farm FLMS, Sibiu county

Analysis index Lactation

Fat SNF Density Protein Lactose pH

I n=60 x±sx 5,27±0,05 9,64±0,03 1,03±0,01 4,90±0,02 4,70±0,04 6,32±0,02

II n=60 x±sx 4,37±0,07 9,42±0,04 1,03±0,01 4,50±0,02 4,65±0,03 6,32±0,02

III n=60 x±sx 4,29±0,08 9,12±0,05 1,033±0,01 3,81±0,03 4,43±0,02 6,30±0,02

IV n=60 x±sx 4,21±0,07 9,14±0,03 1,03±0,01 3,66±0,03 4,23±0,05 6,28±0,02

V n=60 x±sx 4,82±0,09 9,25±0,03 1,03±0,01 3,71±0,04 4,28±0,03 6,25±0,02

VI n=60 x±sx 5,10±0,09 9,42±0,04 1,032±0,01 3,79±0,03 4,39±0,02 6,34±0,02

VII n=60 x±sx 5,49±0,06 9,72±0,03 1,031±0,01 4,20±0,03 4,51±0,03 6,38±0,02

XX  

When the concentrates represents, in the food, 50% of the dry matter of food

intake of goats, mastication and rumination are reduced and the salivation of the rumen

content is decreased. To prevent a decrease of pH in rumen, it is beneficial the presence

of buffer in food, such as sodium bicarbonate and magnesium oxide. Enrichment of food,

within certain limits, with fat (cakes cereals, whey) will lead to the increase of milk

production, fat content and of total protein of such milk (the maximum recorded value

being of 5.49% fat and 4.90% protein), including the amount of casein. Also, the type of

protein in food and their degradability in the rumen, is affecting the milk production, the

fat and protein content of such milk and also its processing properties, particularly in

cheeses.

Fig. 3 The variability of physical-chemical index and average values of goat milk, depending on the lactation stage at the farm FLMS, Sibiu county

Milk protein content can be influenced by the level of energy and protein intake of

the ration. A low energy intake (20-25% of normal), reduces both the total protein

content and the casein fraction of milk. Reducing the protein content of milk during the

first two months of lactation is caused by a lack of energy intake adequate to the

lactation. The milk protein is positively influenced by the presence of slightly soluble

carbohydrates existing in the grain grits, sugar beet, corn and grass green mass. The

increase of the cellulose proportion of the ration leads to the reduced content of protein in

milk, the minimum recorded value being of 3.66% - in the fourth month of lactation

XXI  

(IANCU, 2010). The excess of protein in the ration does not affect milk protein level. A

long time excess of protein (over 30% of normal) leads to a decrease of the casein

fraction content and it increases that of urea in milk. Such milk coagulates when heated.

Table 5

Variability of the physical-chemical index and average values of goat milk,

depending on the lactation stage, at the farm FLH, Vâlcea County

Analysis index Lactation

Fat SNF Density Protein Lactose pH

I n=60 x±sx 5,50±0,05 9,67±0,05 1,03±0,01 4,74±0,03 4,72±0,02 6,29±0,02

II n=60 x±sx 4,82±0,05 9,52±0,03 1,033±0,01 3,92±0,02 4,70±0,04 6,29±0,02

III n=60 x±sx 4,78±0,06 9,44±0,03 1,03±0,01 3,79±0,03 4,43±0,04 6,18±0,02

IV n=60 x±sx 4,52±0,07 9,12±0,03 1,029±0,01 3,58±0,03 4,21±0,05 6,22±0,02

V n=60 x±sx 4,69±0,09 9,14±0,04 1,03±0,01 3,85±0,03 4,21±0,02 6,27±0,02

VI n=60 x±sx 4,98±0,07 9,25±0,04 1,032±0,01 4,22±0,03 4,29±0,02 6,27±0,02

VII n=60 x±sx 5,37±0,06 9,60±0,03 1,033±0,01 4,28±0,03 4,51±0,03 6,37±0,02

Nutritional requirements of lactating goats are the highest as in quality and

quantity level, compared to other periods of the year. Compared with last month of

mammary rest period, in the first month of lactation nutritional requirements increase

from 0.5 to 1.2 times for energy and from 2 to 2.5 times for protein, while the ability to

consume forage in order to ensure this need, remains low.

A goat that produces 3-4 liters of milk per day excretes in milk 120-150 g fat, of

which only a part comes from ingested forage daily. Therefore, in the first month of

lactation goats use their body fat reserves and very little protein reserve. Unlike

pregnancy, this mobilization of fat does not involve the ketonemia risk. To offset the

shortfall of energy, goat’s body may use the metabolism of a quantity of fat, with a yield

XXII  

of 70-75%, to cover daily needs. In the second and third months of lactation nutritional

requirements increase from 1.8 to 2.2 times for energy, than during the mammary rest

period, while the need for protein remains constant (BANU et al., 2007). The explanation

is that fat reserves are exhausted in the first month and the deficit must be covered by the

ration.

0

2

4

6

8

10

I II III IV V VI VII

Valori m

edii ale indicilor 

fizico

‐chimici/The average 

conten

t of p

hysical and

 chem

ical indices

Fază de lactație/Lactation Stage

Grăsime/Fat

SNF

Densitate/Density

Proteină/Protein

Lactoză/Lactose

pH

Fig. 4. Variability of the physical-chemical index and average values of goat milk,

depending on the lactation stage, at the farm FLH, Vâlcea County

The behavior of goat nutrition during the first three months of lactation is based on

the use of hay from vegetables or green mass, with a valuable floristic composition from

the nutritional point of view and a mixture of concentrated whose structure must be

adapted to the obtained production of milk and ingestion capacity. What distinguishes the

goat milk from the cow milk refers to the size of fat globules, which is, on average, 2.5

μm for goat milk and to 2.5 to 3.5 μm for cow milk. It results that the fat globules of goat

milk, which have finer scale, have a greater surface area exposed to the attack of the

lipases and are, therefore, more easily digested compared to that of cow. Also, in goat

milk, the protein responsible for the agglomeration of fat globules doesn’t exist. The

fodder ration for a goat of 50 kg, in the first month of lactation, with a production of 4 kg

of milk/day must provide a of 2.7 kg (alfalfa hay with a dry matter content of 1,28 kg

and concentrated mixture containing 1.04 kg). For the second month of lactation, the dry

substance requirement is 3 kg, which can be composed of: alfalfa-grasses meadow with

1.21 kg D.M., plain hay with 0.17 kg D.M. and mixed concentrates with 0.87 kg D.M.

XXIII  

5.3. CONCLUSIONS

1. To cover the various nutrients in goats, energy and protein supplied by food

must be increased, as these dairy animals have a smaller rumen capacity. Green fodders,

including silage, rich in fiber and/or water are poor/low in energy and protein

components. On the other hand, grain, fat seeds (sunflower, soybeans) contribute to the

increase of the intake energy of goats and the flours from defatted oilseeds increase the

amount of the protein in food. Nutritional, physiological and endocrine status affects

milk production and its composition for shorter or longer periods of time.

2. The milk protein is positively influenced by the presence of slightly soluble

carbohydrates existing in the grain grits, sugar beet, corn and grass green mass. To offset

the shortfall of energy, goat’s body may use the metabolism of a quantity of fat, with a

yield of 70-75%, to cover daily needs.

3. In the second and third months of lactation, nutritional requirements

increase from 1.8 to 2.2 times for energy, than during the mammary rest period, while the

need for protein remains constant. The explanation is that fat reserves are exhausted in

the first month and the deficit must be covered by the ration. A diet with proper

proportion of hay, silage and concentrates as mixed or as pellets or cubes will usually

lead to more milk production, changes in milk fat and protein content, changes that will

affect the manufacture of cheese.

4. The milk production, seasonally limited, can be overcome by hormonal

treatments, but there is a method that does not use hormonal intake: the off season of

breeding. The stimulation or inhibition of inner internal secretion of hormones is made by

regulating the duration of light in the goat’s shelter. Photoperiod is the method that

seasons’ off breeding in goats. The use of light treatments is an alternative to hormonal

treatments for induction and synchronization of heat and ovulation during the period of

seasonally anestrous (infecundity state manifested by the absence of heats).

XXIV  

CHAPTER 6

RESEARCH ON IMPROVING THE NUTRITIONAL QUALITY OF GOAT MILK BY USING VITAMIN B12 SUPPLEMENTS

6.1. MATERIALS AND METHODS

Goat milk is one of the most valuable foods, from a therapeutic standpoint it is the

food with “medicine” qualities. Due to the fineness of fat cells, goat milk is easily

absorbed by intestinal chorionic and thus has a higher digestibility. Under the action of

gastric juice proteins coagulate into fine flakes, are easily digested by photolytic enzymes

of the small intestine and, therefore, are easily assimilated. Goat milk, having a high

content of vitamin A is indicated in the diet of infants, and of the elderly. The knowledge

of the nutritive value of fodders is one of the main issues in the study and practice of the

animals’ nourishment. Animal fodders supply the necessary nutrients for vital functions

and for the achievement of planned production. In light of these considerations it has

been taken into account the supplement of assessing the amount of forage, specifying the

content of substances that satisfies the body requirements on multilateral sides.

Probiotics are substances or food products that are part of our digestive ecosystem

and contain live microorganisms: lactic acid producing bacteria, bifid- and lactic bacteria,

yeasts, which are in the normal intestinal micro biota of a healthy person (IMADA, 2009,

KONGO, 2006; SKEGGS, 1952). A special place in this category it occupies the types of

yeasts found in dairy products that can be used in such probiotic preparations,

(Saccharomyces carlsbergensis).

The field research took place within an animal farm in Sibiu, identified in the

study by the abbreviation FLC and the institutional and organizational framework in

which the research laboratory was made, were: The Center for Research in

Biotechnology and Microbiology of the University Lucian Blaga of Sibiu and the

National Research Institute for Biological Sciences, Bucharest.

The plan of research has been conducted and performed by the following steps:

The first stage to improve the milk quality is the analysis of various dietary supplements

XXV  

rich in vitamin B12. Goat forage ration consisting of alfalfa hay, wheat and oat bran was

enhanced with Saccharomyces carlsbergensis with progressive concentrations, from

1.5% of the total quantity of fodder, up to 3%, considered the second stage. The third

stage is the collection and analysis of milk samples for the determination of vitamin B12

content, prior to the administration of the beer yeast Saccharomyces carlsbergensis f=in

fodder (milk sample identified in the study by the abbreviation LFDN) and after the

administration of the vitamin B12 supplement (milk sample identified in the study with

LCDN abbreviation). The yeast obtained as a byproduct of beer production, dried and

inactivated, can be used as a dietary supplement due to its content of vitamin B. For this

reason, there was made an improvement of goat milk raw material LFDN, respectively

LCDN, with beer yeast flakes Saccharomyces carlsbergensis, representing stage four.

Chromatographic measurements were performed using a HPLC Shimadzu system

with the following components: LC-20AD pumps sp Pump, column Kromasil 100-5C18,

250x4, 6mm (E26265), DGU-20As Degasser, CTO-20AC Column Oven, SPD- M20A

diode array detector, LCMS solution software. Other equipments used to prepare stock

solutions and samples were the analytical balance Mettler Toledo, Heidolph REAX

vortex, Hettich UNIVERSAL320R centrifuge. The mobile phase was filtered through a

membrane CHROMAFIL® O-20/25, 0, 20 and sonic with an ultrasonic bath

TRANSSONIC 460 / H in order to remove the air dissolved in the solution. Samples

were filtered, before injection, through the ultra filtration cells Millex Syringe Driven

Filter Unit, Millipore 0.22 mm. The injection volume for standard vitamin B12, as well as

for the processed samples, was 20 μl. The wavelength to identify the compound of

interest was chosen according to the absorption spectrum, this being 361 nm which is the

specific maximum absorption of cobalamin.

Both standard and samples were analyzed under identical conditions, as the mobile

phase being binary with an A component of heptansulfonic acid (5 mm) in methanol and

a B component consisting of acetic acid (1%) that were filtered through a PTFE

membrane. It was also used a flow rate of 1 ml/minute in isocratic module, the

relationship between the components of the mobile phase being of 30:70 (A: B).

XXVI  

The column was balanced for one hour before injection of the samples. The total

analysis time was 20 minutes; therefore, between two successive analyses the column

was rebalanced with the mobile phase for 20 minutes. After a series of comprehensive

analysis, the HPLC system was cleaned with H2O and methanol for one hour. Analyses

were performed at 250C.

6.2. RESULTS AND DISCUSSION

The determination of vitamin B12 analysis characteristics was performed using

first, standard vitamin B12, Sigma Aldrich. From the analysis of chromatograms it was

observed that the characteristic retention time for vitamin B12 is tR B12= 5.51 min, this

retention time being then used to identify the vitamin B12 in samples, measurements

being made at 361nm. (OPREAN et al., 2010). In order to determine the response with

the variation of the concentration in vitamin B12 a calibration curve was drawn, using

injections of different concentrations of

vitamin B12. Measurements were

performed in triplicate and the calibration

curve is shown in Figure 5. It is noted that

the response showed an excellent linearity

between the peak area characteristic for

vitamin B12 and the concentration of the

injected analyt, over a range of

concentrations between 1.8x10-7  mol /L

- 2.2x10-6 mol/l, the correlation coefficient

being of R2= 0.9976.

Aria 

3.532.521.5Conc ppm10.50

60000

50000

40000

30000

20000

10000

0

y = 18554x - 2262.6R 2  = 0.9976

Fig. 5 Calibration curve for vitamin B12

obtained through the elaborated method

XXVII  

250 300 350 400 nm

0

25

50

75

100

mAU

361

278

306

Fig. 6 Absorption spectrum of

vitamin B12 obtained for one solution

of 100 ppm concentration (Kromasil

column 100-5C18, 250x4,6 mm

(E26265))

The characteristic parameters for vitamin B12 are presented in the table below.

Table 6

Performance characteristics of the chromatographic method developed for the analysis of vitamin B12

Analyt Retention time The straight equation of calibration R2 VitaminB12 tR = 5,51min Y = aX + b

a = 18554 b = -2263

R = 0,9976

The obtained data confirms that the developed method can be used for the

determination of vitamin B12 by high performance liquid chromatography with UV-VIS

detection of the diode-array (DAD) type. Milk samples were analyzed by high

performance liquid chromatography (HPLC) using the method described above, after, in

advance, they have undergone a treatment in order to achieve interferent protein

precipitation and quantitative extraction of vitamin B12 from the sample matrix.

It should be noted that, as stated above, the identification of vitamin B12 was

performed using the specific retention time, obtained for standard, by applying an

acceptable coefficient of variance CV = 0.19 (which leads to an average value of

tR = 6.134 min ± 0.19%).

XXVIII  

20g (±0.02) milk +1,2 g 

Centrifugation, 10min, 1250g, 18oC 

Filtration 0.22um and injection HPLC 

Collection and mixing supernatant fractions brought to 50ml volumetric flask with 4% TCA 

l i

Centrifugation, 10min, 1250g, 18oC 

Residue + 3 ml solution 4% TCA 

Harvesting supernatant 

Fig. 7 Stages of P1 procedure

The samples of milk were processed using two methods of protein precipitation

and extraction of vitamin B12, marked in the text with P1 and P2. A first set of samples of

milk was subjected to the vitamin B12 extraction using trichloroacetic acid (TCA), to

precipitate proteins and to remove the interferences and a second set using acid

hydrolysis (sulfuric acid, SA) (OPREAN et al., 2010). For uniformity, results are

expressed as μg/100 g milk for P1 and for P2 μg/100 ml, according to the steps shown in

Figures 7 and 8. The quantities of vitamin B12 obtained in all samples are shown in

Table 7, where: L **- ACT are the appropriate tests to the first way of processing the

sample, while L **- AS are the samples treated according to the process P2.

As seen from the values presented in Table 7, taking into account the fact that they

are micrograms per 100 g (i.e. 100 ml), the differences between the two extraction

methods exist, but are not dramatic. In addition, it appears that the values adequate for the

concentration of vitamin B12 from the samples present the same type of variation,

regardless the method of processing.

XXIX  

8 ml milk + 45 ml H2SO4 0.1N

Sonicare ultrasonic bath for de 1 h

Fig. 8 Stages of P2 procedure Brought to 100ml flask with methanol

 ‐180CFreezer storage 1 h,

5 min vortexing and filtration through 0.22 um 

HPLC Analysis 

Table 7

Concentration of vitamin B12 in the milk samples using two extraction procedures

No  Sample  Analyt Concentration 

µg/20g Concentration µg/g (ppm) 

Concentration µgB12/100g 

milk 1  LCDNI‐TCA  Vitamin B12  0,1548  0,00774  0,7740 2  LCDN 1‐TCA  Vitamin B12  0,3694  0,01847  1,6491 3  LCDN 2‐TCA  Vitamin B12  0,7169  0,03584  3,5844 4  LCDN 3‐TCA  Vitamin B12  1,0999  0,05499  4,9725 5  LFDNI‐TCA  Vitamin B12  0,1499  0,00749  0,6850 6  LFDN 1‐TCA  Vitamin B12  0,4402  0,02201  2,0504 7  LFDN 2‐TCA  Vitamin B12  0,7236  0,03618  3,6178 8  LFDN 3‐TCA  Vitamin B12  1,0427  0,05213  4,5652 9  LCDNI ‐AS  Vitamin B12  0,1835  0,00917  2,2933 10  LCDN 1‐AS  Vitamin B12  0,1883  0,00941  2,3533 11  LCDN 2‐AS  Vitamin B12  0,2833  0,01416  3,5417 12  LCDN 3‐AS  Vitamin B12  0,3022  0,01511  3,7775 13  LFDNI ‐AS  Vitamin B12  0,1312  0,00656  1,6398 14  LFDN1‐AS  Vitamin B12  0,2308  0,01154  2,8848 15  LFDN2‐AS  Vitamin B12  0,2688  0,01344  3,3605 16  LFDN3‐AS  Vitamin B12  0,3370  0,01685  4,2120 

XXX  

Chromatograms for results illustrating are:

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 min

-250

0

250

500

750

1000

1250

uV

Fig. 9 Chromatography obtained for vitamin B12 at 3 levels of

concentration on the calibration curve (0, 75; 1, 5 and 3 ppm)

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000uV

Fig. 10 Chromatography obtained for the initial LCDNI milk samples, with yeast addition: LCDNI-blue,

LCDN1-red, LCDN2-black, LCDN3-green, (column Kromasil 100-5C18, 250x4, 6mm (E26265))

4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 min0

500

1000

1500

2000

2500

3000

3500

4000

uV

Fi lk

L -

g. 11 Chromatograms obtained for misamples with added initially LFDN yeast:

FDN-red, one black LFDN, LFDN 2green-blue LFDN 3

XXXI  

Both the yeast Saccharomyces carlsbergensis and fodder have undergone

extraction processes like the milk samples, but being left to macerate for 12h. The content

of vitamin B12 from yeast, as well as the one from the used forage, is qualitively

noticeable, but in amounts below the linearity range established for the method, as shown

in Figures 12 and 13.

5.0 6.0 7.0 8.0 min

1

2

3

4

5

6

7

8

9

10

11

12

13

14

mAU

1/vitaminaB

12

5.0 7.5 10.0 min

-10

0

10

20

30

40

50

60

70

80

90

100

mAU

1/vita

minaB

12

Fig. 12 Chromatography obtained

for the fodder sample

Fig. 13 Chromatography obtained for the Saccharomyces carlsbergensis.

XXXII  

6.3. CONCLUSIONS

1. In general, vitamins are synthesized by the animal organism only in a very

small measure, being mainly synthesized by plants and microorganisms. Animal and

human bodies get their vitamins from food, through diet, being in a free state or as

inactive, of pro-vitamins, which are converted later in the body into active vitamin, after

some physical-chemical or biochemical reactions. In the animal kingdom and at humans,

the presence of vitamins along the substances with plastic and energetic role is absolutely

necessary, because the partial or complete lack of them causes serious disorder, which if

not corrected it may result in the death of the body. Vitamins are required by their bio-

catalyze action of the metabolic processes, reason for which they are given attention in

the complex assessment of forage. Although they are only needed in small amounts

(up to 40 mg / kg D.M.), vitamins are indispensable for life.

2. From this study it was found that yeast has some advantages over bacteria,

in their use as recombinant microorganisms with probiotic role: they are easy to cultivate,

are the first complete genome sequence of eukaryotic (Saccharomyces carlsbergensis), in

the yeast cell may happen necessary changes for the expression of some proteins (e.g.

proteolysis, glicolysis), are not sensitive to most commonly used antibiotics (e.g.

penicillin), presents “in vitro” a higher survival rate in the stomach and small intestine.

3. Due to the important role in nutrition and relative instability of vitamins,

qualitative and quantitative analysis is an important goal for manufacturers and food

processors. High performance liquid chromatography (HPLC) is the preferred analytical

technique for determining vitamins solving many of the complex analytical problems that

arise in analyzing real samples and can be applied after the extraction of the target

analytes in solid phase. The imbalance between food intake and diet necessities causes

profound metabolic changes, especially because the human body is increasingly

demanded by environmental pollution and many stress factors.

XXXIII  

GENERAL CONCLUSIONS

The main quality of milk is the food or nutritional value and is even greater as it

respond to the body needs. Based on the optimal results from my own situations and from

the literature data, the nutritional and hygienic quality of goat milk has improved.

Analyzing the results and also the risks that may occur in the raw milk, from the

primary source of production (farms), we would like to highlight a few conclusions:

1. The determination of the quality of goat milk produced by different

production systems regarding the influence of specific pathogens from milk:

staphylococcus, streptococcus and Escherichia coli reveal that the highest rate of intra-

mammary infection was recorded for semi-intensive production system. In certain

circumstances, the use of milking facility is responsible for the spread of bacteria on

nipple canal. This could explain why Staphylococcus aureus was identified only in the

cases when it was used the container milking installation. This observation indicates that

the method of milking influences the health of the udder.

2. The analysis of the hygienic quality indicators regarding the count

evaluation of somatic cell shows us that in order to identify the udder’s infection it is also

necessary the comparison or correlation with the existing number of bacterial cells, such

as total number of bacterial cells, the number of staphylococcus or the number of coli

form bacteria. The number of pathogenic microorganisms from milk can then be a

quantitative index of inflammation of the mammary gland.

3. Monitoring of physic-chemical quality of goat milk from the two different

geographical areas was done according to season and stage of lactation. It was noted that

within the same species and breed, the lactation period has a strong influence on the

composition of milk. As for goats, both colostral milk and that from the first period of

lactation is richer in fat and protein, then these components decrease and then increase at

the end of the lactation period, when the milk production is lower. Major and minor

components in milk depend on the season, but compositional differences may be

influenced by the type of feeding. The milk production, seasonally limited, can be

XXXIV  

overcome by hormonal treatments, but there is a method that does not use hormonal

intake: the off season of breeding. The stimulation or inhibition of inner internal secretion

of hormones is made by regulating the duration of light in the goat’s shelter. Photoperiod

is the method that seasons’ off breeding in goats. The use of light treatments is an

alternative to hormonal treatments for induction and synchronization of heat and

ovulation during the period of seasonally anestrous.

4. The processing of the experimental data was done with statistical means

starting with the gathering, grouping of the observed data as a preparatory stage regarding

the application of specific statistical methods and techniques. The obtained analytical

data were processed using modern statistical means applied in a computerized system. As

a statistical model it was used F test (Fisher) - ANOVA test. At the statistical process of

the obtained data was primarily done the calculation of the following statistical

parameters: arithmetic average, deviation, standard and relative standard deviation.

For the interpretation of the obtained analytical results, it was proceeded to analyze the

statistical significance of differences between the environments studied using F-test

(Fischer) at probability levels P * 0.05, statistical calculations were performed with

ANOVA Sigle Factor calculation algorithm (Microsoft Excels).

5. The Determination of the vitamin B12 in animal food was made: for the

supplement of their nutritional value in order to meet the requirements of the animal

organism and to increase the amount of vitamin B12 in raw milk. The vitamin B12 is found

in the goat milk in an amount up to four times greater than in the cow milk.

6. From the research on the vitamin B12 content in different dietary

supplements it has been found that yeast has some advantages over bacteria, in their use

as recombinant microorganisms with probiotic role: they are easy to cultivate, are the first

complete genome sequence of eukaryotic (Saccharomyces carlsbergensis).

Saccharomyces carlsbergensis yeast obtained as a byproduct of beer production, dried

and inactivated by mechanical ways, can be used as a dietary supplement due to its

content of vitamin B12 (containing 0.015 μg, 1.5% of the Recommended Daily

Allowance).

XXXV  

7. The assessment of the nutritional quality of goat milk raw material

enhanced with vitamin B12 supplements was achieved by chromatographic

measurements, using a Shimadzu HPLC system. The first unconventional method of

improving nutritional quality of milk was the administration of the yeast Saccharomyces

carlsbergensis in forage. Following the obtained results, in the raw milk was found a

growth of vitamin B12 as a percentage of 12.99% compared to the blank. The second

method consisted of adding the Saccharomyces carlsbergensis yeast flakes in different

proportions (5, 10, 15 g/100 ml milk), directly in the raw milk. The resulted quantity of

vitamin B12 in milk was higher than for the first method, respectively with : 199.32% for

5 g/100 ml milk, 428.14% for 10 g/100 ml milk and 566.45% for 15 g/100 ml of milk.

8. The B group vitamins are relatively resistant to pasteurization temperatures

(are destroy at about 10 ... 20%), excepting vitamin B12, which its quantity is essentially

decreased. Sterilization destroys vitamin B12 up to 90% of the total quantity of raw

material and it is considerably reduced at the preservation of milk in the light.

 

XXXVI  

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XXXVII  

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