research of the production process of tangerine marmalade and tangerine jam

Research on the ratio of added sugar for the production of tangerine marmalade .... Study on the effect of sugar mix ratio on product quality in tangerine marmalade .... Processing diagr

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURAL AND FORESTRY

HOANG THỊ LAN OANH

RESEARCH OF THE PRODUCTION PROCESS OF TANGERINE MARMALADE AND TANGERINE JAM

BACHELOR THESIS

Study Mode: Full-time

Faculty: International Programs Office

Thai Nguyen, 2/01/2021

i

ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Environmental Science and Management Student name Hoàng Thị Lan Oanh

ii Keywords: Tangerine, Tangerines Jam, Tangerines Marmalade,

Tangerine Products

Date of Submission: 14/12/2021

ACKNOWLEDGEMENT

First of all, I want to thank you for all the support you have given me during my 4 years at Thai Nguyen University of Agriculture and Forestry, Advanced Education Program I studied, and researched my full-time internship program at the Faculty of Biotechnology and Food Technology

I would like to express my deep gratitude to Master Pham Bang Phuong who enthusiastically instructed and created the best conditions for me to complete this graduation thesis He pointed out the errors, the nonconformities, and how to fix them during the time here Once again I sincerely thank my supervisor, and wishing good health for him

Besides, I have received a lot of interest, and help from Mr Nguyen Van Quang and my team who interns at the laboratory of the Faculty of Biotechnology – Technology and Technology for helping me throughout the project implementation I hope that my team will always unite together to solve all difficult things I wish you guys will find your path, and succeed on the path you choose

Given the limited time as well as experience, the mistake of the thesis cannot be avoided I look forward to receiving the guidance, and comments of the teachers so that I can supplement, improve our knowledge, and better serve the practical word in the future

PART II LITERATURE REVIEW 7

2 The scientific basis of the thesis 7

2.1 Tangerine Overview 7

2.1.1 Tangerine peel and tangerine juice overview 7

2.1.2 Feature of tangerine juice and tangerine peel 8

2.1.3 The chemical composition and nutritional value of tangerine juice 8

2.2 Overview of jam products 10

2.2.1 Jam Citrus products 10

2.2.2 General process of producting tangerine jam and tangerine marmalade 11

2.2.2.1 General process of producting tangerine jam 11

2.2.2.2 General process of producting tangerine marmalade 12

2.3 Tangerine production of some northern provinces in Vietnam 12

PART III CONTENT AND METHODS 14

3.1.3.1 Food safety assessment based on national standards 14

3.1.3.2 Analyze the composition and indicators of product concern 14

3.1.3.3 Evaluation of quality variation of jam and marmalade products from tangerine, preserved under normal conditions 14

3.2 Research methods 15

3.2.1 Survey of demand and sensory evaluation of tangerine jam and tangerine marmalade 15

3.2.2 Approach method and experimental layout 15

3.2.2.1 Research on the ratio of added sugar for the production of tangerine jam 17

3.2.2.2 Research on the ratio of added sugar for the production of tangerine marmalade 18

3.2.2.3 Study on the ratio of Cyclo Dextrin to the ability to reduce bitterness of tangerine marmalade 18

3.2.2.4 Study on the ratio of Citric Acid added to tangerine jam 19

3.2.2.5 Study on the ratio of Citric Acid added to tangerine marmalade 19

3.2.2.6 Study on the ratio of pectin affecting the structure of tangerine jam 19

3.2.2.7 Study on the ratio of pectin affecting the structure of tangerine marmalade 203.2.2.8 Study on the ratio of gelatine to the structure of tangerine jam 20

3.2.3 Food safety assessment of microbiological contamination limits 21

3.2.3.1 Determination of microorganisms in food according to TCVN 4884-2:2015 21

3.2.3.2 Quantitative method of Escherichia coli according to TCVN 7924-1:2008 223.3 Analysis of quality parameters of tangerine jam and tangerine marmalade products 263.3.1 Determination of moisture (loss in mass at 103 oC) (based on TCVN 9741: 2013) 26

3.3.2 Method for determination of total ash (based on TCVN 9742: 2013) 28

3.3.3 Method of determining vitamin C content (according to TCVN 4715:1989) 30

3.3.4 Method of determining reducing sugar content (according to TCVN 4075:2009) 32

3.3.5 Evaluation of changes in quality of soluble products of tangerines stored under normal conditions 34

3.3.6 Sensory evaluation method according to TCVN 3215 – 79 34

vii

PART IV RESULTS 38

4.1 Survey results on consumer demand for Tangerine jam and Tangerine marmalade 38

4.2 Analysis of research and improve processing of products 41

4.2.1 Analysis of research and improve processing of Tangerine Jam 42

4.2.1.1 Study on the effect of sugar mix ratio on product quality in tangerine jam 42

4.2.1.2 Study on the effect of the ratio of Citric Acid on the ability to adjust taste in tangerine jam 42

4.2.1.3 Study on the effect of Pectin ratio on the structure of tangerine jam 43

4.2.1.4 Study on the influence of Gelatine ratio on the structure of tangerine jam 44

4.2.2 Analysis of research and improve processing of Tangerine Marmalade 45

4.2.2.1 Study on the effect of sugar mix ratio on product quality in tangerine marmalade 45

4.2.2.2 Research on optimizing the ratio of Cyclo Dextrin to the ability to remove bitterness at normal temperature 46

4.2.2.3 Study on the effect of the ratio of Citric Acid on the ability to adjust taste in tangerine marmalade 47

4.2.2.4 Study on the effect of Pectin ratio on the structure of tangerine marmalade 474.2.3 Perfecting the production process of tangerine jam and tangerine marmalade 494.2.3.1 Completing the production process of tangerine jam 49

4.2.3.2 Completing the production process of tangerine marmalade 53

4.3 Product quality analysis results 56

4.3.1 Microoganisms results of tangerine Jam and tangerine Marmalade 56

4.3.2 Evaluate the nutritional quality of the tangerine jam and the tangerine marmalade 57

PART V DISCUSSION AND CONCLUSION 59

5.1 Discussion 59

5.2 Conclusion 59

5.2.1 Survey on consumer tastes and preferences for tangerine jam and tangerine marmalade products 60

5.2.2 Improving procuction process for tangerine jam and tangerine marmalade 60

5.2.2.1 Developing production process for tangerine jam products 60

5.2.2.2 Developing a production process for tangerine marmalade products 60

5.2.3 Quality assessment of jam and marmalade from tangerine 61REFERENCES 62APPENDICES 63

1

LIST OF FIGURES

Figure 1 General process of producting tangerine jam 11

Figure 2 General processing of producting marmalade 12

Figure 3 Processing diagram of Tangerine Jam 16

Figure 4 Processing diagram of Tangerine Marmalade 17

Figure 5 Results of a survey on consumer demand for fruit jam products in Thai Nguyen 38

Figure 6 The flow chart of the production process of tangerine jam 49Figure 7 The flow chart of the production proces of tangerine marmalade 53

LIST OF TABLES

Table 1: Tangerines producing countries in the world 4Table 2 Nutritional composition in 100g of tangerine juice 8Table 3 Research on the ratio of added sugar for the production of tangerine jam 17

Table 4 Research on the ratio of added sugar for the production of tangerine marmalade 18

Table 5 Study on the ratio of Cyclo Dextrin to the ability to reduce bitterness of tangerine marmalade 18

Table 6 Study on the ratio of Citric Acid added to tangerine jam 19Table 7 Study on the ratio of Citric Acid added to tangerine marmalade 19

Table 8 Study on the ratio of pectin affecting the structure of tangerine jam 19

Table 9 Study on the ratio of pectin affecting the structure of tangerine marmalade 20

Table 10 Study on the ratio of gelatine to the structure of tangerine jam 20

Table 11 Product quality level according to sensory quality scores 34Table 12 Distribution of weight coefficients for sensory parameters 35Table 13 Sensory scoring facility 35Table 14 The effect of Gelatine ratio on the structure of tangerine jam 44Table 15 Effect of sugar ratio on sensory quality of tangerine marmalade 45

Table 16 Effect of the ratio of Cyclo Dextrin on the anti-bitterness of tangerine marmalade 46

Table 17 Effect of the ratio of Citric Acid on the ability to adjust taste 47

3

Table 18 Effect of pectin ratio on the structure of tangerine marmalade 48Table 19 Microorganisms in the product 57Table 20 Product quality analysis results 57

PART I INTRODUCTION 1 Research rationale

In the world, the genus Citrus has about 20-25 species, distributed in East, South and Southeast Asia, Australia, the southwest Pacific islands, with many varieties growing throughout the country with a harmonious climate In Vietnam, the genus Citrus has 15 species, mostly grown, including tangerines

Tangerine is the general name only with a number of the same genus Citrus Vietnam, South China and Japan are considered the birthplace of these tangerine tangerine varieties by people and found wild relatives in this area, as in China Said has been cultivated for at least 3,000 years now and is widely cultivated to get results in many tropical to tropical regions The cultivation of tangerines produces an output of about 33 million, of which 6 countries have an output of 1 million tons or more (Table 1)

Table 1: Tangerines producing countries in the world

No Countries Fruit production in 2017 (million tons)

5 Tangerines with sugar, small fruit, thick skin, very sweet, are grown in Phu Tho, Tuyen Quang, Yen Bai, Hanoi, ; Tangerines, small fruits, thick and sour skin, are grown mainly in the mountains with an altitude of 800-1,600m in Lang Son, Ha Giang, Lai Chau, There are many other varieties imported and planted in Tangerines such as pink tangerines, green tangerines, Thai tangerines,

In Vietnam, tangerines are grown mainly for fruit, essential oil distillation and medicine In Traditional Medicine, many parts are used differently such as: Ripe pods (Bare skin), peel of green pods (Serum), outer shell of fruit (Kumquat), Leaves (Quat), seeds (Quat ganglion)

Tangerine peel (Tran derm) is a part widely used in traditional medicine in Vietnam, China, The main ingredient of Tran vera is essential oil with the main ingredients D-limonene, and flavonide, including hesperidin, methylhesperidin, nobiletin, tangeretin, are the active ingredients of most interest in the dermis

In tangerine juice contains a lot of Vitamin C, it contains 36 mg of vitamin C in each fruit Vitamin C is good for your hair, skin, immune system and weight balance, and it's also a great vitamin for your digestive system Vitamin C helps regulate the intestines and improves the absorption of nutrients from other foods In addition, tangerines have only 35 calories in each fruit They contain free fats and antioxidants, which can help slow down the aging process

Tangerine seeds have a bitter taste, calculated in average, in 2 canals and kidneys; has the function of gas, hemolysis, and system; used to treat intestinal prolapse, scrotal pain, inflammation of the mammary glands, back pain with a dose of 3-9g / day

Tangerine leaves have a spicy taste, bitterness, in the two canals and waste, the function of the first, gas, phlegm; used to treat chest pain; cough, breast swelling, intestinal prolapse at a dose of 10-20g/day

Vietnamese tangerines are cheap and have a short harvest time So it's difficult to consume, the value is low Tangerine products are few, not diverse Jam and Marmalade in Vietnam market do not yet have, especially tangerine products On that basis, I conducted this research in order to diversify products from

tangerines, improve the value of tangerine products, help tangerine growers have a stable income, not dependent on the seasons

2 Research’s objectives

2.1 Overall objectives

Research and improve technology in the production of tangerine jams, take advantage of and solve the problem of improving the value of tangerine in the province by taking advantage of cheap and affordable tangerine raw materials, diversifying products tangerine products

- In terms of economy and society:

The successful implementation project will be the basis for the initial production of tangerine jam and tangerine marmalade and transfer the production process to local people to diversify products from tangerines, thereby increasing income and improving life

2.1 Tangerine Overview

2.1.1 Tangerine peel and tangerine juice overview

Tangerine is a mandarin orange variety called after Tangier, Morocco, where it originated While there are many different types of tangerines available today, they are all called mandarin orange hybrids Because of their intense, sweet flavor, tangerines are a popular citrus fruit They have a reputation for being sweeter than oranges in general As a result, they're a tasty and healthful dessert alternative Tangerines, like all citrus fruits, are high in vitamin C Tangerines also contain a moderate amount of vitamin A, with 100 grams providing around 14% of your daily necessary vitamin A consumption Eating tangerine peels has also been linked to health advantages Tangerine is a super-flavonoid, or antioxidant, found in the peel In studies, super-flavonoids have showed promise as a means to decrease cholesterol

Having enough vitamin C in your body has been linked to having good skin Vitamin C has been demonstrated to play an important function in the production of collagen in the body Collagen is the substance that gives our skin its youthful appearance Vitamin C has also been demonstrated to help prevent and repair skin damage from the sun Another advantage of tangerines' high vitamin C content is that it promotes eye health by preventing cataracts and age-related macular degeneration Vitamin C and other nutritional antioxidants have been shown to help keep your eyes healthy for longer

Tangerine peel is taken by mouth for a variety of reasons, including asthma, indigestion (dyspepsia), cancer prevention, and many others, although there is no clear scientific evidence to back up these claims Tangerine fruit and peel are both edible Tangerine juice can also be prepared from the fruit

2.1.2 Feature of tangerine juice and tangerine peel

It can provide various health benefits to the body in addition to giving a wide range of key vitamins and characteristics Tangerine is a left-handed tree with few qualities and a complete fat You will be challenged on portion sizes in your primary meals because the process of eating will give you a feeling of hunger rapidly and for a long time There is a wide range, much like fruits, with plenty of vitamin C In fact, just one serving can provide up to 26% of your daily vitamin C requirements Vitamin C is important for a variety of reasons, the most important of which is its impact on immune function

Getting enough vitamin C each day can help lessen the symptoms of common respiratory infections like colds and minimize their duration Not only that, but a lack of this essential vitamin can lead to lowered resistance to illnesses and infections One of the most notable tangerine advantages is its high antioxidant content, which includes naringin, naringenin, nobelitin, narirutin, and hesperidin Antioxidants are potent molecules that aid in the battle against the detrimental effects of free radicals in the body, lowering the risk of inflammation and chronic diseases such as cancer and diabetes

Cough, nausea, constipation, and flatulence can all be relieved with tangerine peel Repel foul breath and improve sleep quality

2.1.3 The chemical composition and nutritional value of tangerine juice

Table 2 Nutritional composition in 100g of tangerine juice

Citrus juice is a good source of flavonoids, which have a variety of biological benefits, including antioxidant properties and the ability to treat cancer Dietary flavonoids, in particular, operate on carcinogenic active chemicals, activate carcinogen detoxification, remove free radicals, govern cell cycle progression, limit cell proliferation, and so forth Preclinical research has shown that these juices have

anticancer properties in both in vitro and in vivo settings (lab animals)

2.2 Overview of jam products

Tangerine juice: It has vibrant colors and a pleasant flavor, and it's made from freshly squeezed tangerine juice that's been pasteurized to prevent spoiling

Tangerine syrup is mostly used in the production of soft foods and beverages, but it can also be used to make ice creams, dairy goods, bread products, ready meals, jams, and other syrups

Tangerine Essential Oil is made from the peel of various Citrus Sinensis fruits and is 100% natural This product was created by a mechanical process that used a cold-pressed method with no thermal modifications Then, immediately after extraction, pack it in drums to preserve its qualities

2.2.1 Jam Citrus products

Fruit puree is a product made from fresh fruit or semi-prepared (fruit puree, fruit juice, sulphated fruit ) cooked with sugar to 65-70% dryness, with the addition of pectin or agar-agar to create frozen gel Jam products stand out in the sweet taste and characteristic aroma of the fruit Processing jam is also one of the important measures to preserve fresh fruit The product is completely different from the starting material in terms of usage In the world, fruit puree has been applied in production and trade with ingredients such as strawberries, cherries, blueberries, pineapples, papayas But the products of tangerine puree just stop at manual processing, serving the needs of the family, there has not been any specific research on the production of tangerine puree in the direction of application at the production scale

Marmalade prepared from fruit (fresh, sulfite or frozen) whole or cut, cooked with sugar, with or without added food acids and pectin

Jelly is a type of jam whose main ingredient is fruit juice instead of fresh fruit like jam, then added sugar and pectin, etc to make jam Therefore, jelly is usually clearer and smoother than jam and can use herbs, tea, etc even wine to make jelly instead of fruit

11

2.2.2 General process of producting tangerine jam and tangerine marmalade

2.2.2.1 General process of producting tangerine jam

Figure 1 General process of producting tangerine jam

2.2.2.2 General process of producting tangerine marmalade

Figure 2 General processing of producting marmalade

2.3 Tangerine production of some northern provinces in Vietnam

Bac Son yellow tangerine is famous for its sunny yellow color, sweetness and aroma, giving the tangerine a special flavor Currently, the total area of tangerine trees in the district is over 600 hectares, of which the area for harvesting is over 400 hectares, the tangerine production of the cooperative in recent years has reached

13 about 185 tons

Trang Dinh tangerine is also a highly appreciated tangerine variety, it has the characteristics of large fruit, large and succulent segments, straw yellow shrimp, sour and sweet taste The largest tangerine growing area in Trang Dinh district is more than 280 hectares The yield of the 2020 tangerine crop is estimated at 2.2 tons/ha, 4 quintals/ha higher than in 2019

Tra Linh tangerine (Cao Bang) has long been known for its sweet taste and distinctive aroma that no other drink has Up to now, in Tra Linh district, there are 164 hectares of tangerine trees, the yield is about 12-15 tons/ha

In Bac Kan province, the entire area of oranges and tangerines is around 3,500 hectares, with around 2,400 ha harvested Bac Kan oranges and tangerines are expected to produce around 24,500 tons in 2021, with tangerines accounting for more than 17,700 tons and oranges accounting for roughly 6,800 tons.

PART III CONTENT AND METHODS 3.1 Content

3.1.1 Survey of demand and sensory assessment of tangerine Jam and Marmalade tangerine products

Conduct surveys on consumption and demand for jam products

3.1.2 Research and improve processing of jam and marmalade products from

3.1.3 Quality assessment of jam and marmalade from tangerine

3.1.3.1 Food safety assessment based on national standards

Products are assessed for microbiological contamination limits in food according to QCVN 8-3: 2012 / BYT - National technical regulation for microbiological contamination in food The product is determined microorganisms in food according to TCVN 4884-2: 2015 and quantitative Escherichia coli according to TCVN 7924-1: 2008

3.1.3.2 Analyze the composition and indicators of product concern

The product is determined moisture, sugar, dissolved dry matter, vitamim, total ash according to national standards

3.1.3.3 Evaluation of quality variation of jam and marmalade products from tangerine, preserved under normal conditions

1 Determination of microorganisms in tangerine jam products

2 Quantification of Escherichia coli bacteria in tangerine jam products 3 Determination of moisture content of tangerine jam products

4 Determination of total ash in tangerine jam products

15 5 Determination of vitamin C content in tangerine jam products

6 Determination of reducing sugar content in tangerine jam products

7 Evaluation of changes in quality of tangerine jam products preserved under normal conditions

3.2.2 Approach method and experimental layout

Over the past years, tangerines have been widely used by consumers, many people have expanded cultivation and tending tangerines according to technical processes, bringing into play the value of bringing income to the people However, harvesting tangerines is only seasonal, with a short harvest time (2-3 months), in many places the cost is lower Therefore, to increase economic value for tangerines and tangerine products, group research and apply science and technology to processing and diversifying tangerine products to increase income, increase value and increase income for people

Jam products from tangerine are produced according to the following general process:

Citrus raw materials are processed without seeds, film processing materials and pressed to collect tangerine juice The juice is inhibited and removed from the citrus-bitter ingredients group (Limonin, nomilin, naringin…) Tangerine juice and a part of the untouched tangerine meat are used in different recipes to cook onions and produce tangerine jam Tangerine jam is researched and produced according to the technological process described in Figure 1

Figure 3 Processing diagram of Tangerine Jam

Marmalade products from tangerines are produced according to the following general process:

Citrus raw materials are processed seedless, raw materials processing and pressed to collect tangerine juice The juice is inhibited and removed from the citrus-bitter ingredients group (Limonin, nomilin, naringin…) Tangerine extract and a part of tangerine peel are used in different recipes to cook and produce tangerine Marmalade Tangerine Marmalade is researched and manufactured according to the technological process described in Figure 2

17

Figure 4 Processing diagram of Tangerine Marmalade

3.2.2.1 Research on the ratio of added sugar for the production of tangerine jam

Table 3 Research on the ratio of added sugar for the production of tangerine jam

Evaluate the sensory quality of each product and choose the right sugar mixing ratio

3.2.2.2 Research on the ratio of added sugar for the production of tangerine marmalade

Table 4 Research on the ratio of added sugar for the production of tangerine marmalade

19

3.2.2.4 Study on the ratio of Citric Acid added to tangerine jam

Table 6 Study on the ratio of Citric Acid added to tangerine jam

Assess the sensory quality of each product and choose the appropriate citric acid mixing ratio

3.2.2.5 Study on the ratio of Citric Acid added to tangerine marmalade

Table 7 Study on the ratio of Citric Acid added to tangerine marmalade

Assess the sensory quality of each product and choose the appropriate citric acid mixing ratio

3.2.2.6 Study on the ratio of pectin affecting the structure of tangerine jam

Using the research results, we can mix the ratio of ingredients with fixed factors as follows:

+ Ratio of citric acid: 0.05% + Ratio of sugar to juice: 60%

Table 8 Study on the ratio of pectin affecting the structure of tangerine jam

Evaluate the sensory quality of each product and choose the most appropriate ratio

3.2.2.7 Study on the ratio of pectin affecting the structure of tangerine marmalade

Using the research results, we can mix the ratio of ingredients with fixed factors as follows:

+ Ratio of citric acid: 0.05% + Ratio of sugar to juice: 60%

Table 9 Study on the ratio of pectin affecting the structure of tangerine marmalade

Evaluate the sensory quality of each product and choose the most appropriate ratio

3.2.2.8 Study on the ratio of gelatine to the structure of tangerine jam

Using the research results, we can mix the ratio of ingredients with fixed factors as follows:

+ Ratio of citric acid: 0.05% + Ratio of sugar to juice: 50% + Pectin ratio: 0.6%

Table 10 Study on the ratio of gelatine to the structure of tangerine jam

21

3.2.3 Food safety assessment of microbiological contamination limits

3.2.3.1 Determination of microorganisms in food according to TCVN 2:2015

4884 Rule:

A specified quantity of the test sample, or a specified amount of the initial suspension in the case of other products, is poured onto the surface of a solid agar culture medium in a Petri dish

Other dishes are prepared under the same conditions, using a decimal dilution of the test sample or the initial suspension

Plates were incubated aerobically at 30 °C for 72 h

Calculate the number of microorganisms per gram or milliliter of test sample by the number of colonies obtained in dishes containing less than 300 colonies

- Proceed:

Test portion, initial suspension and dilutions

As specified in ISO 6887 or the specific standard relevant to the product concerned

+ Inoculation and incubation

Using a sterile pipette, transfer 0.1 ml of the test sample if the product is liquid or the initial suspension (10-1 dilution in the case of other products) to the center of the two agar plates If plates from more than one dilution are prepared, they can be reduced to one plate [TCVN 6404 (ISO 7218)]

For some products, it is preferable to count small numbers of microorganisms, the limit of detection can be increased by a factor of 10 by inoculating 1.0 ml of the test sample if the product is liquid or 1.0 ml of the sample initial suspension of the product in another form, on the surface of one large agar plate (140 mm) or on the surface of three small agar plates (90 mm) In either case, prepare dual plates using two large agar plates or six small agar plates

Get another jelly plate Using another sterile pipette dispense 0.1 ml of 10-1 diluent (liquid products) or 0.1 ml of 10-2 diluents (other products)

If necessary, repeat the procedure with subsequent decimal dilutions, using a new sterile pipette for each decimal dilution

Carefully spread the inoculum as quickly as possible over the surface of the agar plate, not allowing the array to touch the side of the plate If possible, use the same set of samples for all dilutions from a sample with the highest dilution,

Leave the plates covered for about 15 min at ambient temperature for the inoculum to absorb into the agar

Turn the prepared dishes upside down and place in the incubator (6.3) set at 30 °C in accordance with ISO 7218 Incubate for (72 ± 3) h

NOTE Using a spiral inoculation device for culture, see Annex A + Counting colonies

After the specified incubation period, retain plates with less than 300 colonies, if possible Count the colonies on the plates, using a colony-counting device, if necessary Colonies must be counted correctly to avoid miscounting food particles with major colonies

Spreading colonies are considered as single colonies If growth of colonies is expected, examine the plates after 24 h or 48 h and mark visible colonies If colonies grow to less than one-quarter of the plate, count the colonies on the remainder of the plate and calculate the corresponding number for the whole plate If the colony grows to more than one-quarter of the plate, the plate should not be counted If all plates have spreading colonies, count the most appropriate plates and record in the report that the results may be influenced by spreading colonies

3.2.3.2 Quantitative method of Escherichia coli according to TCVN 1:2008

7924 Rule:

Inoculate a specified amount of the test sample or a specified amount of the initial suspension onto cellulose membranes coated on modified mineral glutamate agar (MMGA), then incubate at 37 °C for 4 h

Use decimal dilutions of the test sample or of the initial suspension inoculated

23 in two dishes for each dilution, under the same conditions

For isolation, transfer membranes from the recovery phase on MMGA to tryptone-bile-glucuronide (TBX) agar, and then incubate at 44 °C for 18 h to 24 h

The number of colony-forming units (CFUs) of β-glucuronidase-positive Escherichia coli per gram or per milliliter of sample is calculated from the number of typical green CFUs

- Proceed:

Test portion, initial suspension and subsequent dilutions

See ISO 6887-1 or the specific standard relevant to the product to be determined

+ Recovery

Using the sterile blunt end clamps (6.6), place the cellulose acetate membrane (6.5) on the dry surface of the two MMGA agar plates (5.2.1.3), taking care not to mix air bubbles into the filter membrane Use a sterile spreader (6.12) to flatten the membrane, if necessary

Using a sterile pipette (6.8), place 1 ml of the test sample or initial suspension in the center of the membrane Using a sterile spreader, spread the inoculum evenly over the membrane surface, avoiding spillage from the membrane

Repeat the procedure in 9.2.1 with further decimal dilutions, if necessary, using another sterile pipette and a different sterile spreader for each diluent

Leave the inoculated plates horizontally at room temperature for about 15 min until the inoculum has penetrated deeply into the agar Incubate the plates for 4 h ± 1 h in the incubator (6.2) set at 37 °C with the membrane/agar face up

+ Switch to selective medium and incubate

After recovery, using sterile clamps, transfer membranes from MMGA (recovery medium) to agar plates containing TBX medium

WARNING – The wet film will adhere to the agar surface Avoid air bubbles Do not use the sampler set

Incubate the plates for 18 h to 24 h in an incubator set at 44 °C and not more than 45 °C, with the membrane/agar side facing up Do not stack discs more than three layers

Counting of colony forming units (CFU)

After the specified incubation period, count typical CFUs of glucuronidase-positive Escherichia coli on each agar plate containing less than 150 typical CFU and less than 300 total CFU (typical and atypical)

β-If the inoculum cannot be separated and typical colonies cannot be observed, dishes containing typical 0 CFU should be considered according to different calculation methods as in clause 10

- Show results

+General requirements

Calculations should take into account the most common cases when carried out in accordance with good laboratory practice There are also some exceptional but rare cases (e.g very different CFU numbers between two plates from the same diluent, or much different ratios from a single dilution factor between different diluents) plates from two consecutive dilutions) Counting results should be checked and interpreted by a qualified analyst, and the results may also be rejected

N = (1) Inside

Σa is the sum of colonies counted on all plates retained after two consecutive

25 dilutions, with at least one plate containing at least 15 blue CFUs;

n1 is the number of plates retained at the first dilution; V volume of inoculum used per plate, in millilitres; n2 is the number of plates retained at the second dilution;

d the dilution factor corresponding to the first dilution is retained [d=1 in the case of (liquid samples) directly inoculated test specimens]

Round the results to two significant figures (see ISO 7218:2007)

Take as a result the number of β-glucuronidase-positive Escherichia coli per milliliter (liquid products) or per gram (other products) expressed as whole numbers to two significant figures (less than 100) or as words 1.0 to 9.9 times powers of 10

+ Estimate small numbers

If two dishes [of the test sample (liquid product) or of the initial suspension (other product) or of the first dilution are inoculated or retained] containing less than 15 typical CFUs, calculate NE number of CFUs of β-glucuronidase-positive Escherichia coli present in the test sample is the mean of the parallel plate according to Equation (2):

NE = (2) Inside

Σc is the sum of typical CFUs counted on the two plates; V volume of inoculum used per plate, in millilitres; n is the number of disks retained (in this case n = 2);

d the dilution factor corresponding to the first dilution is retained [d = 1 in the case (liquid samples) when the test sample is inoculated directly]

Round the results to two significant figures (see ISO 7218:2007) The results are displayed as follows:

- Estimated number of β-glucuronidase-positive Escherichia coli per

milliliter (liquid product) or per gram (other product): NE = Y

If the two dishes [of the test sample (liquid product) or the initial suspension (other product) or from the inoculated or retained first dilution] contain no typical CFU, express the result as after:

- Less than 1/d of β-glucuronidase positive Escherichia coli per milliliter (liquid products) or per gram (other products);

Where d is the dilution factor of the initial suspension or the first dilution inoculated or retained [d = 1 when using the liquid product (test sample) without dilution]

If all typical or atypical CFUs on two dishes at the first dilution d1 contain more than 300 CFUs, the blue color is visible, or if on two dishes at the next dilution d¬2 contains less 300 colonies, with no typical CFU to be counted, express the result as follows:

- Less than 1/d2 and more than 1/d1, β-glucuronidase-positive Escherichia coli present in one milliliter (liquid products) or per gram (other products);

Where d1 ¬ and d2 are the dilution factors for diluents d1 and d2, respectively

If all typical or atypical CFUs on two dishes at the first dilution d1 contain more than 300, no typical CFUs are visible, or if on two dishes at the next dilution d2¬ contains less than 300 colonies, with no typical CFU to be counted, express the result as follows:

- less than 1/d2 of β-glucuronidase-positive Escherichia coli CFUs per milliliter (liquid products) or per gram (other products);

Where d2 are the dilution factors corresponding to the dilution d2

3.3 Analysis of quality parameters of tangerine jam and tangerine marmalade products

3.3.1 Determination of moisture (loss in mass at 103 oC) (based on TCVN 9741: 2013)

- Sampling according to TCVN 9743:2013

27 - Principle: Dry the soluble tea test portion in an oven at 103 °C for 2 h and weigh the dried test portion

NOTE The drying time is approximately 2 h because this period gives the best results compared to those obtained using the Karl Fischer method for the determination of the moisture content of instant tea

- How to proceed

+ Prepare weighing box or weighing cup chén

Open the lid of the weighing box or crucible and dry the container or crucible with the lid for 1 h in a temperature controlled oven at 103 °C ± 2 °C Cool in a desiccator After cooling to room temperature, close the lid and weigh to the nearest 0.001g

+ Sample section

Open the sample container and immediately weigh approximately 4g of the prepared test sample, to the nearest 0.001 g, into the prepared weighing box or crucible, with the lid on the side

+ Determine

Dry the weighing crucible or container with the test portion, opening the lid and leaving the lid next to it immediately in the oven set at (103 °C ± 2 °C) to constant mass (usually for 4 h) The cabinet door should not be opened during the drying period

Open the oven and immediately break the lid of the weighing pan or box Transfer to a desiccator, open the lid (leave the lid in the desiccator) and cool in a covered desiccator for at least 30 min Remove the weighing crucible from the flask, close the lid, and weigh to the nearest 0.001 g Determine the mass of the dry test portion to the nearest 0,001 g

+ Number of times determined

Carry out two determinations on the same test sample + Show results

Moisture or mass loss at 103 °C, expressed as a percentage by mass of the sample, is given by the formula:

For moisture content in the range 3% (m/m) to 4% (m/m), the difference between the results of two determinations, carried out consecutively (or simultaneously) by the same operator , using the same apparatus, on the same test piece, shall not exceed 0,2% (absolute value)

+ Reproducibility

For moisture content in the range 3% (m/m) to 4% (m/m), the difference between the final results obtained by two laboratories using this method to analyze the same laboratory sample , not more than 0.8% (absolute value)

3.3.2 Method for determination of total ash (based on TCVN 9742: 2013)

- Principle: Decompose organic matter by treating with concentrated hydrochloric acid solution and heating at 550 oC Weigh the rest

- Proceed:

+ Prepare the cup

Ensure that the crucible is completely clean, then heat for at least 30 min at 550 °C ± 25 °C in the furnace Cool in a desiccator After cooling to room temperature, weigh to the nearest 0.001 g

29 + Sample section

Open the sample container and immediately weigh approximately 2 g of the prepared test sample, to the nearest 0.001 g, into the prepared crucible Spread the sample evenly over the bottom of the cup

+ Determine

Add the concentrated hydrochloric acid solution dropwise to the test portion in the crucible, using sufficient quantity (approximately 1 ml) to completely moisten the sample

Place the crucible on a cooled electric stove, turn it on to medium and cook for 30 min Raise the hob temperature to the highest setting in three consecutive steps, at each step, cook for 30 min Hold the test portion at the highest heating until no smoke appears for at least 5 min

Place the crucible containing the test portion in the furnace at 550 °C ± 25 °C for 16 h Remove, let cool and add a few drops of water to moisten and disperse the ash Evaporate to dryness on a hotplate as above and return to the furnace and heat for a further 30 min Remove, cool to room temperature in a desiccator, and weigh to the nearest 0.001 g Determine the mass of total ash to the nearest 0.001 g

NOTE:

+ Number of times determined

Carry out two determinations on the same test sample + Show results

Total ash, expressed as a percentage by mass of dry matter, is calculated using the following formula:

Inside

m0 is the mass of the test portion, in grams (g); m1 is the total mass of ash, in grams (g);